Peptide fragment condensation and cyclisation using a subtilisin variant with improved synthesis over hydrolysis ratio

ABSTRACT

The invention relates to a method for enzymatically synthesizing an (oligo)peptide, comprising coupling (a) an (oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptide nucleophile having an N-terminally unprotected amine, 
     wherein the coupling is carried out in a fluid comprising water, and 
     wherein the coupling is catalyzed by a subtilisin BPN′ variant or a homologue thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof : 
     a deletion of the amino acids corresponding to positions 75-83; 
     a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221 selenocysteine; 
     preferably a mutation at the amino acid position corresponding to P225 wherein the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2. 
     Further, the invention relates to an enzyme suitable for use as a catalyst in a method of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The invention is a divisional of and claims priority to U.S. application Ser. No. 15/517,812, filed on Mar. 26, 2015, which is a national stage entry of PCT/NL2015/050711 filed Oct. 9, 2015, which claims priority to PCT/NL2015/050441, filed Jun. 16, 2015, which claims priority to PCT/NL2014/050707, filed Oct. 10, 2014, the entire contents of each of which are hereby incorporated in total by reference.

BACKGROUND

The invention relates to a method for enzymatically synthesising an (oligo)peptide (i.e. a peptide, in particular oligopeptide), to an enzyme suitable for catalyzing said synthesis, to a host cell capable of functionally expressing said enzyme and to a method for preparing said enzyme. Peptides, in particular oligopeptides have many applications, for instance as pharmaceutical, food or feed ingredient, or cosmetic ingredient.

Methods for synthesizing (oligo)peptides are generally known in the art.

Oligopeptides can be chemically synthesized in a stepwise fashion in solution or on the solid phase via highly optimized processes. However, peptides longer than 10-15 amino acids are often very difficult to synthesize due to side reactions and as a consequence purification is troublesome. Therefore, peptides longer than 10 amino acids are often synthesized by a combination of solid-phase synthesis of side-chain protected oligopeptide fragments which are subsequently chemically condensed in solution, e.g. as in a 10+10 condensation to make a oligopeptide of 20 amino acids. The major drawback of chemical side-chain protected oligopeptide fragment condensation is that upon activation of the C-terminal amino acid residue of the acyl donor racemisation occurs. In contrast, enzyme-catalyzed peptide couplings are completely devoid of racemisation and have several other advantages over chemical peptide synthesis such as the absence of side reactions on the side-chain functionalities. For industrial application, an enzymatic peptide synthesis concept based on a kinetic approach, i.e. using an acyl donor C-terminal ester is most attractive (see for instance N. Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology”, 1^(st) reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002).

Chemo-enzymatic peptide synthesis can entail the enzymatic coupling of oligopeptide fragments which have individually been synthesized using chemical synthesis, fermentation, or by a combination of chemical and enzymatic coupling steps. Some reports have been published on the enzymatic condensation of oligopeptide fragments in aqueous solution (Kumaran et al. Protein Science, 2000, 9, 734; Björup et al. Bioorg. Med. Chem. 1998, 6, 891; Homandberg et al. Biochemistry, 1981, 21, 3387; Komoriya et al. Int. J. Pep. Prot. Res. 1980, 16, 433). However, a major drawback of such enzymatic oligopeptide fragment condensation in aqueous solution is that simultaneous hydrolysis of the peptide bonds within the oligopeptide fragments and of the C-terminal ester functionality takes place leading to low yields and many side products.

Proteases have hitherto mainly been produced commercially for hydrolytic application, e.g. in cleaning, where peptide bonds are hydrolysed by the proteases. A typical example are the subtilisins, which form an enzyme class with considerable importance for their use as detergents. Therefore, subtilisins have been the subject of numerous protein engineering studies. subtilisins have also been used for the synthesis of oligopeptides, which was, however, almost always accompanied by hydrolytic side-reactions to a significant extent. It was found by Wells et al. (U.S. Pat. No. 5,403,737) that the condensation of oligopeptides in aqueous solution could be significantly improved by altering the active site of subtilisin BPN′, a subtilisin from B. amyloliquefaciens (SEQUENCE ID NO: 2). When two mutations were introduced, i.e. S221C and P225A, a subtilisin BPN′ variant called subtiligase was obtained having a 500-fold increased synthesis over hydrolysis ratio (S/H ratio) as compared to wild-type subtilisin BPN′. However, the average ligating yield was around 66% and hydrolysis of the oligopeptide acyl donor C-terminal ester was still substantial (Wells et al. Science, 1994, 266, 243). Most often, 10 equivalents of oligopeptide acyl donor C-terminal ester was used to obtain a decent reaction yield. Another drawback of subtiligase was the poor stability against organic co-solvents that are required to solubilize the oligopeptide fragments, against enhanced temperature and against denaturating agents, which are often needed for successful oligopeptide condensation. Therefore, Wells et al. added five additional mutations to subtiligase, i.e. M50F, N76D, N109S, K213R and N218S, to make the enzyme more stable (Proc. Natl. Acad. Sci. USA, 1994, 91, 12544). The new mutant called stabiligase appeared moderately more resistant to sodium dodecasulfate and guanidinium hydrochloride, but hydrolysis was still a major side reaction. For instance an oligopeptide carboxyamidomethyl-ester (Cam-ester) was ligated to an oligopeptide amine using stabiligase in a yield of 44%. In this example, 10 equivalents of the oligopeptide C-terminal ester were used and thus, 9.56 equivalents of the oligopeptide C-terminal ester were hydrolyzed at the C-terminal ester functionality and only 0.44 equivalents ligated to the oligopeptide amine to form the product. Clearly, there is a need for an improved enzyme with a higher S/H ratio to make the oligopeptide condensation reaction an economically viable process. Probably for this reason, the past 20 years subtiligase nor stabiligase have been industrially applied, to the best of the inventors knowledge.

Another aspect of subtilisin BPN′ that has received attention is the increase of the stability of the enzyme for its use as detergent (i.e. for the hydrolysis of peptide bonds) at higher temperatures and/or in the presence of metal chelators. A typical example of such study was disclosed by Bryan et al. who engineered a subtilisin BPN′ variant lacking a high affinity Ca²⁺ binding site (WO02/22796). The high affinity Ca²⁺ binding site in subtilisin BPN′ is made up by a loop comprising amino acids 74-82 and the amino acids Gln2 (Q2) and Asp41 (D41). Comparison of the 3D structure of subtilisin BPN′ with the structure of homologous subtilisins shows that the high affinity Ca²⁺ binding site is highly conserved. This binding site is important for their stability in known subtilisins. Stripping of the Ca²⁺ ion by for instance metal chelators leads to unfolding and thus inactivation of the known subtilisins. When Bryan et al. deleted amino acids 75-83 (Δ75-83) of subtilisin BPN' and additionally implemented the mutations Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, T254A and Q271E, a subtilisin BPN′ variant was obtained (called BS149, also known as Sbt149) which lacks the Ca²⁺ binding domain and has a greatly improved stability against metal chelators (1000x). However, this enzyme cannot be used for peptide fragment condensation in aqueous solution since it is only hydrolytically active.

It is an object of the present invention to provide an enzymatic method for preparing an (oligo)peptide by condensation of a first and a second (oligo)peptide fragment or by cyclisation of an (oligo)peptide that can serve as an alternative to known methods of preparing (oligo)peptides. There is a need for alternative methods in general, in particular in order to broaden the palette of tools for making specific (oligo)peptides.

In particular, it is an object to provide an enzymatic method for preparing an (oligo)peptide by condensation of a first and a second (oligo)peptide fragment or by cyclisation of an (oligo)peptide, wherein an enzyme is used having an improved S/H ratio and stability compared to subtilisin BPN', at least under certain reaction conditions.

Further, it is an object to provide an enzymatic method for preparing an (oligo)peptide by condensation of a first and a second (oligo)peptide fragment or by cyclisation of an (oligo)peptide, wherein an enzyme is used having an improved stability compared to subtiligase, in particular an improved stability and S/H ratio compared to subtiligase.

It is yet a further object of the invention to provide the coupling of a (oligo)peptide to a protein. It is in particular a challenge to provide enzymatic methodology that allows coupling of a peptide with a protein, in particular due to the added complexity of a protein's three-dimensional structure.

It is yet a further object to provide a novel subtilisin BPN′ variant capable of catalyzing the condensation of two (oligo)peptides or of cyclisation of an (oligo)peptide, in particular such an enzyme having an improved property, such as an improved synthesis over hydrolysis ratio ratio and/or improved stability, compared to known enzymes suitable to catalyse such condensation, such as subtilisin BPN′ and/or subtiligase, at least under certain reaction conditions.

One or more other objects that may be subject of the invention follow from the description below.

It has now surprisingly been found that it is possible to provide a subtilisin BPN′ variant wherein the calcium binding domain at the positions corresponding to amino acids 75-83 has been inactivated, namely by deletion, that has catalytic activity with respect to the condensation of two (oligo)peptide fragments or the cyclisation of a peptide, and in particular to provide such a variant that has an improved S/H ratio compared to subtilisin BPN′ and/or subtiligase, by providing a subtilisin BPN' variant that has a specific mutation, preferably a specific combination of mutations, in addition to the deletion of the amino acids corresponding to positions 75 to 83.

Accordingly, the present invention relates to a method for enzymatically synthesizing an (oligo)peptide, comprising coupling (a) an (oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptide nucleophile having an N-terminally unprotected amine,

wherein the coupling is carried out in a fluid comprising water, and

wherein the coupling is catalyzed by a subtilisin BPN' variant or a homologue thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof:

i) a deletion of the amino acids corresponding to positions 75-83;

ii) a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221selenocysteine (S221U);

iii) preferably, a mutation at the amino acid position corresponding to P225;

wherein the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.

Further, the invention relates to a method for enzymatically synthesizing a cyclic (oligo)peptide of at least 12 amino acids, comprising subjecting an (oligo)peptide C-terminal ester or thioester having an N-terminally unprotected amine to a cyclisation step wherein said cyclization is carried out in a fluid comprising water, and

wherein the cyclization is catalyzed by a subtilisin BPN′ variant or a homologue thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof:

i) a deletion of the amino acids corresponding to positions 75-83;

ii) a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221selenocysteine;

iii) preferably, a mutation at the amino acid position corresponding to

P225;

wherein the amino acid positions are defined according to the sequence of subtilisin BPN' represented by SEQUENCE ID NO: 2.

Further, the invention relates to an enzyme, which enzyme is a subtilisin BPN′ variant or homologue thereof, comprising the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or homologue sequence thereof:

i) a deletion of the amino acids corresponding to positions 75-83;

ii) a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221selenocysteine;

ii) a mutation at the amino acid position corresponding to P225;

in which the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.

Further, the invention relates to a recombinant method for preparing the enzyme according to the invention, said method comprising:

-   -   a) providing a recombinant host cell functionally expressing a         gene encoding the enzyme;     -   b) culturing said host cell under conditions which provide for         the expression of the enzymatically active enzyme; and     -   c) recovering the expressed enzyme from said microbial host.

Further, the invention relates to a recombinant polynucleotide comprising a sequence which encodes for an enzyme according to the invention.

Further, the invention relates to a host cell, comprising a polynucleotide according to the invention. The host cell is capable of functionally expressing the enzyme of the invention.

Further, the invention relates to the use of an enzyme according to the invention as a catalyst. Such use generally comprises contacting one or more substrates (reactants) in the presence of the enzyme under conditions wherein the enzyme catalyses a chemical reaction wherein the substrate(s) participate(s). The enzyme has been found particularly useful as a catalyst in peptide synthesis. It is in particular contemplated that an enzyme of the invention is useful for catalyzing reactions of which known subtilisins are known to be catalytically active. In an embodiment, the synthesised peptide is a protein. In an embodiment the synthesised peptide is an oligopeptide. In a further embodiment the synthesised peptide is composed of at least 201 amino acid units.

The invention provides a useful alternative to known methods of preparing (oligo)peptides, including proteins extended with an (oligo)peptide.

Moreover, it has surprisingly been found possible with a method of the invention to enzymatically condense two (oligo)peptide fragments or to cyclize an (oligo)peptide in a liquid comprising water with a high synthesis over hydrolysis ratio. The method of the invention is advantageous in that it offers the possibility for coupling various oligopeptide fragments in aqueous solution in high yield without substantial hydrolytic side reactions. Such surprising finding is illustrated by the Examples, which show that a method of the invention is not only suitable to synthesise (oligo)peptides that lack a secondary and tertiary protein structure, but also allows coupling two peptide fragments wherein at least one of the fragments is a protein, thereby synthesizing an (elongated) protein provided with an additional sequence of amino acid units. It has been found possible to synthesise such protein whilst maintaining a secondary and tertiary structure of the protein.

For the purpose of this invention, with “synthesis over hydrolysis ratio” (S/H ratio) is meant the amount of enzymatically synthesised (oligo)peptide product divided by the amount of (oligo)peptide C-terminal ester or thioester of which the ester or thioester group has been hydrolysed.

The value of the S/H ratio of an enzyme of the invention depends on various factors, for instance the nature of the substrates (the amino acid sequences of the (oligo)peptide C-terminal ester or thioester and of the (oligo)peptide nucleophile) and reaction conditions (e.g. temperature, pH, concentration of the peptide fragments, enzyme concentration). As shown in the Examples, it was found though that under various reaction conditions and for various substrates the S/H ratio was higher than for known subtilisins, such as subtiligase and subtilisin BPN′. Thus, it is contemplated that the S/H ratio of an enzyme according to the invention in general has a significantly higher S/H ratio than subtiligase and subtilisin BPN′, when tested under the same reaction conditions and using the same substrates, and in particular it is contemplated that an enzyme of the invention has a significantly higher S/H ratio under the conditions used in Example 1 (100 mM phosphate buffer, pH 8.0, temperature about 20° C., concentration of (oligo)peptide C-terminal ester 0.83 mM, concentration of (oligo)peptide nucleophile 3.33 mM, enzyme concentration 5.5 mg/L) or one or more of the other examples. Thus, in particular, the invention relates to a subtilisin BPN′ variant or homologue thereof wherein the S/H ratio of the subtilisin BPN′ variant or homologue thereof divided by the S/H ratio of subtiligase—at least under the conditions described in Example 1 or one or more of the other Examples—is more than 1, preferably 2 or more, in particular 5 or more. The upper value of this quotient is not critical; in practice it may e.g. be 100 or less, in particular 20 or less.

The S/H ratio of the subtilisin BPN' variant or homologue thereof of the invention divided by the S/H ratio of subtilisin BPN′—at least under the conditions described in Example 1 or one or more of the other Examples—is usually more than 100, preferably 250 or more, more preferably 500 or more, in particular 1000 or more. The upper value of this quotient is not critical; The S/H ratio of subtilisin BPN′ at least under the reaction conditions specified herein is generally very low, it may be even zero (no detectible synthesis). Thus, the S/H ratio of the subtilisin BPN′ variant or homologue thereof of the invention divided by the S/H ratio of subtilisin BPN′ may approximate infinity. In a potential circumstance wherein subtilisin BPN′ has substantial ligase or cyclase activity, the inventors consider that the S/H ratio of the subtilisin BPN′ variant or homologue thereof of the invention divided by the S/H ratio of subtilisin BPN′ is also high, e.g. up to 100 000, in particular up to 25 000, more in particular up to 10 000.

Further, using a method of the invention, the (oligo)peptide product is very easy to purify from the reaction mixture because only little hydrolytic by-products are formed.

Another advantage of the invention is that, due to the improved S/H ratio, a small or no excess of the (oligo)peptide C-terminal ester or thioester or of the (oligo)peptide nucleophile is needed to reach a high yield (>80%) in the condensation reaction. Accordingly, in an advantageous embodiment an (oligo)peptide C-terminal ester or thioester and an (oligo)peptide nucleophile are contacted in a small excess of one of said (oligo)peptide fragments or in an about stoichiometric ratio although a larger excess of one over the other may be used, as described below.

As illustrated by the Examples, an enzyme according to the invention is also advantageous in that it allows the synthesis of a cyclic (oligo)peptide with significantly higher yield than with subtiligase (78% versus 61% for subtiligase). Cyclic (oligo)peptides are a particularly interesting class of peptides since they are often more potent due to their more constrained three dimensional structure and higher resistance to proteolysis.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show enzymatic activity respectively S/H ratio of different enzymes of the invention, compared to subtiligase; all indicated mutations on M222, Y104, I107 and/or L135 are additional to those of BS149-DM. The name ‘BS149-DM’ is used herein for the subtilisin BPN′ variant which has the following mutations compared to subtilisin BPN′ (SEQUENCE ID NO 2): a deletion of the amino acids 75-83 (Δ75-83) S221C , P225A, Y217L, Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A and Q271E.

FIGS. 2A and 2B show activity respectively S/H ratio of different BS149-DM+M222P+L217 mutants; all indicated mutations on L217 are additional to those of BS149-DM+M222P.

FIG. 3A: The P4 pocket specificity of BS149-DM and BS149-DM+Y104 mutants.

FIG. 3B: The P4 pocket specificity of BS149-DM and BS149-DM+I107 mutants.

FIG. 3C: The P4 pocket specificity of BS149-DM and BS149-DM+L135 mutants

FIG. 4A: The P1′ pocket specificity of BS149-DM and BS149-DM+M222A, M222E and M222Q mutants

FIG. 4B: The P2′ pocket specificity of BS149-DM and BS149-DM+M222A, M222E and M222Q mutants

FIG. 4C: The P1′ pocket specificity of BS149-DM and BS149-DM+M222G, M222N and M222P mutants

FIG. 4D: The P2′ pocket specificity of BS149-DM and BS149-DM+M222G, M222N and M222P mutants

FIG. 5A: The P1′ pocket specificity of BS149-DM+M222P+L217N, L217T and L217E mutants

FIG. 5B: The P2′ pocket specificity of BS149-DM+M222P+L217N, L217T and L217E mutants

FIG. 5C: The P1′ pocket specificity of BS149-DM+M222P+L217I, L217V and L217A mutants

FIG. 5D: The P2′ pocket specificity of BS149-DM+M222P+L217I, L217V and L217A mutants

FIG. 5E: The P1′ pocket specificity of BS149-DM+M222P+L217M, L217K and L217Q mutants

FIG. 5F: The P2′ pocket specificity of BS149-DM+M222P+L217M, L217K and L217Q mutants

FIG. 5G: The P1′ pocket specificity of BS149-DM+M222P+L217S, L217G and L217Y mutants

FIG. 5H: The P2′ pocket specificity of BS149-DM+M222P+L217S, L217G and L217Y mutants

FIG. 5I: The P1′ pocket specificity of BS149-DM+M222P+L217F, L217H and L217W mutants

FIG. 5J: The P2′ pocket specificity of BS149-DM+M222P+L217F, L217H and L217W mutants

FIG. 5K: The P1′ pocket specificity of BS149-DM+M222P+L217R, L217C, L217D and L217P mutants

FIG. 5L: The P2′ pocket specificity of BS149-DM+M222P+L217R, L217C, L217D and L217P mutants

FIG. 6A: The P1′ pocket substrate specificity of BS149-DM+M222G+L217N, L217T and L217E mutants

FIG. 6B: The P1′ pocket substrate specificity of BS149-DM+M222G+L2171, L217V and L217A mutants

FIG. 6C: The P1′ pocket substrate specificity of BS149-DM+M222G+L217M, L217K and L217Q mutants

FIG. 6D: The P1′ pocket substrate specificity of BS149-DM+M222G+L217S, L217G and L217Y mutants

FIG. 6E: The P1′ pocket substrate specificity of BS149-DM+M222G+L217F, L217H and L217R mutants

FIG. 6F: The P1′ pocket substrate specificity of BS149-DM+M222G+L217C, L217D and L217P mutants

FIG. 7A: The P1′ pocket specificity of BS149-DM, BS149-DM+M222G and BS149-DM+I107V+M222G mutants

FIG. 7B: The P2′ pocket specificity of BS149-DM, BS149-DM+M222G and BS149-DM+I107V+M222G mutants

FIG. 7C: The P4 pocket specificity of BS149-DM, BS149-DM+I107V and BS149-DM+I107V+M222G mutants

FIG. 8: S/H ratio of BS149-DM+M222G mutant at different pH values

FIG. 9A: S/H ratio of BS149-DM+M222G mutant using different concentrations of acyl donor and H-Glu-Leu-Arg-NH₂ nucleophile

FIG. 9B: S/H ratio of BS149-DM+M222G mutant using different concentrations of acyl donor and H-Ala-Leu-Arg-NH₂ nucleophile

FIG. 10: S/H ratio of different enzymes of the invention used for (oligo)peptide cyclization, compared to subtiligase.

FIG. 11: S/H ratio of BS149-DM+M222G mutant used for (oligo)peptide cyclization at different pH values

FIG. 12: B. subtilis/E. coli shuttle vector pBE-S with BS149-DM gene (pBES DNA-BS149-DM HIStag)

FIG. 13: B. subtilis/E. coli shuttle vector PBS42-S5 with Subtiligase gene

FIG. 14: list of subtilisins that may be used as a template for the provision of homologues of subtilisin BPN′ variants of the invention and the alignment of the sequence segments containing the Ca2+ binding loop with the corresponding loop in subtilisin BPN′ (SEQ ID NO 2) and the deletion of the loop in BS149-DM (SEQ ID NO 5).:

DESCRIPTION

The polynucleotide of the invention can be in either single or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.

The recombinant polynucleotide of the invention is typically synthetic. The invention in particular extends to DNA or RNA isolated from any organism. In a specific embodiment, the invention extends to a host cell comprising recombinant DNA according to the invention. The host cell is typically transgenic.

The term “recombinant” as used herein, refers to a polynucleotide or a cell containing the polynucleotide, which is the result of one or more genetic modifications using (a) recombinant DNA technique(s) and/or (an)other mutagenic technique(s). In particular a recombinant cell may comprise a polynucleotide not present in a corresponding wild-type cell, which polynucleotide has been introduced into that cell using recombinant DNA techniques (a transgenic cell), or which polynucleotide not present in said wild-type cell is the result of one or more mutations—for example using recombinant DNA techniques or another mutagenesis technique such as UV-irradiation—in a polynucleotide sequence present in said wild-type cell (such as a gene encoding a wild-type polypeptide, such as an enzyme) or wherein the polynucleotide sequence of a gene has been modified to target the polypeptide product (encoding it) towards another cellular compartment. Further, the term “recombinant (cell)” in particular relates to a strain (cell) from which DNA sequences have been removed using recombinant DNA techniques.

In particular, the introduction of a mutation into a polynucleotide sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Furthermore mutated genes may be obtained by gene synthesis which apart from the introduction of changes at the amino acid level, may also be used to optimize the coding sequence to improve transcription and translation (R. Carlson, Nature Biotechnology, 2009, 27, 1091; E. Angov et al., PLoS ONE 2008, 3(5): e2189.

The term “transgenic cell ” as used herein, refers to a strain (cell) containing a polynucleotide not naturally occurring in that strain (cell) and which has been introduced into that strain (cell) using recombinant DNA techniques, i.e. a recombinant cell).

The term “or” as used herein is defined as “and/or” unless it is specified otherwise or it follows from the context that it means ‘either . . . or . . . ’.

The term “a” or “an” as used herein is defined as “at least one” unless it is specified otherwise or it follows from the context that it should refer to the singular only.

When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included, unless it follows from the context that it should refer to the singular only.

For the purpose of this invention, with “peptides” is meant any chain composed of two or more amino acids. Thus, peptides are generally amides at least conceptually composed of two or more amino carboxylic acid molecules (i.e. amino acids) by formation of a covalent bond from the carbonyl carbon of one to the nitrogen atom of another with formal loss of water. The term is usually applied to structures formed from alpha-amino acids. A peptide may be linear, branched or cyclic. A peptide can have a single chain composed of two or more amino acids or a peptide can have a plurality of chains. In the case a peptide is composed of two or more chains, each chain generally is composed of three or more amino acid molecules. The amino acid sequence of a peptide is referred to as the primary structure.

In an embodiment, the peptide is essentially free of a secondary structure and essentially free of a tertiairy structure.

In a further embodiment, the peptide has a secondary structure. Secondary structures are generally highly regular local sub-structures, such as alpha-helices and beta-sheets (or beta-strands), by interactions between the individual amino acids and the peptide backbone.

In an embodiment, the peptide (or plurality of peptides) has a tertiary structure. Tertiary structures are generally formed by multiple interactions, among others hydrogen bonding, hydrophobic interactions, van der Waals interactions, ionic interactions and disulphide bonds. The secondary structure can also contribute to the tertiary structure. The tertiary structure provides a three-dimensional shape (which is essentially fixed in a stable environment, such as in the absence of a change in temperature and in the absence of a change in the medium wherein the peptide is present, etc.). As the skilled person knows, the tertiary structure is different from a random coil peptide chain lacking any fixed three-dimensional structure. Proteins are (oligo)peptides having a tertiary structure. A well known example of tertiary structure is the globular structure of globular proteins. In an embodiment, the protein is a protein for target delivery of a pharmaceutically active (oligo)peptide to a specific site, e.g. to a tumour or to organ tissue. Well known examples of proteins, suitable for such purpose, are immunoglobulins or parts thereof, such as an antigen-binding fragment (Fab) of an immunoglobulin. Immuglobulins coupled to a pharmaceutically active (oligo)peptide can thus be used to more efficiently deliver a pharmaceutically active (oligo)peptide to a target, e.g. tumor tissue or organ tissue, that contain an antigen for the immunoglobulin. In an embodiment, the protein is a protein suitable to increase the half-life of an (oligo)peptide in a living organism, in particular the blood plasma half-life. Albumins are examples of proteins that can be coupled to an (oligo)peptide to increase the half-life.

Disulphide bonds (disulphide bridges) are typically bonds between two cysteine units (formed by oxidation). Thus, two amino acids in a same peptide chain (amino acid sequence) can be covalently bound, also if they are not adjacent amino acids in the amino acid sequence. Also, a disulphide bond between a first cysteine of a first peptide chain and a second cysteine of a second peptide chain, which may have the same or a different amino acid sequence, can be formed to form a peptide. Such peptide comprises more than one peptide chain. An example of a peptide composed of more than one peptide chain, wherein the different chains are bound via a disulphide bond is insulin. Other bonds to join different peptide chains are generally known in the art.

In an embodiment, the (oligo)peptide essentially consists of amino acid units. In a further embodiment, the (oligo)peptide essentially consists of amino acid units and protective groups. In an embodiment, the peptide is a conjugate of a peptide chain of two or more amino acids and another molecule, in particular a carobohydrate or a lipid. These peptides are called glycopeptides and lipopeptides respectively. In a further embodiment, the peptide conjugate is a conjugate of two or more amino acids and an imaging agent, such as a fluorescent, phosphorescent, chromogenic or radioactive group. The peptide conjugate may also contain a chelation agent or toxin.

Typically, a peptide—which term includes oligopeptides, proteins and peptide conjugates—comprises up to about 35 000 amino acid units, in particular 3-20 000 amino acid units, more in particular 4-5 000 amino acid units, preferably 5-1000 amino acid units. In a specifically preferred embodiment the peptide comprises 500 amino acid units or less, in particular 200 or less, more in particular 100 or less In a specifically preferred embodiment, the peptide comprises at least 10 amino acid units, more specifically at least 15 amino acids, at least 25 amino acids or at least 40 amino acids.

With “oligopeptides” is meant within the context of the invention, a peptide composed of 2-200 amino acid units, in particular composed of 5-100 amino acid units, more in particular composed of 10-50 amino acid units.

The term “(oligo)peptide” is used herein as a short-hand for the phrase “peptides, in particular oligopeptides”.

The (oligo)peptide that is synthesized may be linear, branched or cyclic. Good results have been achieved with the synthesis of a linear or cyclic oligopeptide. Further good results have been achieved in the synthesis of a peptide having more than 200 amino acid units, e.g. of about 800 amino acid units. Thus, the peptide can have at least 250 amino acid units or at least 400 amino acid units. Further, good results have been achieved with the coupling of a peptide fragment to a protein, such as insulin, whilst maintaining a secondary and tertiary protein structure. The protein can have 200 or less amino acid units or can have more than 201 amino acid units.

The non-cyclic (oligo)peptides are synthesized from a first (oligo)peptide and a second (oligo)peptide, which are both smaller than the (oligo)peptide that is synthesized. The first (oligo)peptide is an (oligo)peptide C-terminal ester or thioester and the second (oligo)peptide comprises an N-terminally unprotected amine. The (oligo)peptide C-terminal ester or thioester is also referred to as an (oligo)peptide acyl donor. The second (oligo)peptide is also referred to as an (oligo)peptide nucleophile. These (oligo)peptides from which the synthesised (oligo)peptide is formed are referred to herein as ‘(oligo)peptide fragments’. These (oligo)peptide fragments can on their turn be synthesized enzymatically from a smaller (oligo)peptide acyl donor and an (oligo)peptide nucleophile or by regular chemical solution or solid phase peptide synthesis, known by the person skilled in the art.

-   -   For the purpose of this invention, with “peptide bond” is meant         the amide bond between (i) either the alpha-amino terminus of         one alpha-amino acid or the beta-amino acid terminus of one         beta-amino acid and (ii) either the alpha-carboxyl terminus of         one other alpha-amino acid or the beta-carboxyl terminus of one         other beta-amino acid. Preferably, the peptide bond is between         the alpha-amino terminus of one amino acid and the         alpha-carboxyl terminus of another amino acid.     -   For the purpose of this invention, with “cyclic peptide” is         meant an (oligo)peptide chain wherein the alpha-amino terminus         and the alpha-carboxyl terminus of a branched or linear         (oligo)peptide are linked via a peptide bond, thereby forming a         ring structure of at least 12 amino acid units. The cyclic         peptide is in particular composed of 12-200 amino acid units,         more in particular composed of 12-100 amino acid units and         preferably composed of 12-50 amino acid units.     -   For the purpose of this invention, with “condensation” is meant         the formation of a new peptide bond between the C-terminal         carboxylic function of an (oligo)peptide with the N-terminal         amine function of another (oligo)peptide or of the same         (oligo)peptide.

In the context of this application, the term “about” means in particular a deviation of 10% or less from the given value, more in particular 5% or less, even more in particular 3% or less.

As defined by Schechter and Berger, the active site residues in proteases, including subtilisins, are composed of contiguous pockets termed subsites. Each subsite pocket binds to a corresponding residue in the peptide substrate sequence, referred to here as the sequence position. According to this definition, amino acid residues in the substrate sequence are consecutively numbered outward from the cleavage sites as . . . -P4-P3-P2-P1-P1′-P2′-P3′-P4′- . . . (the scissile bond is located between the P1 and P1′ positions), while the subsites in the active site are correspondingly labelled as . . . -S4-S3-S2-S1-S1′-S2′-S3′-S4′-. (Schechter and Berger, Biochem Biophys Res Commun. 1967 Apr. 20; 27(2):157-62.)) .

-   -   For the purpose of this invention, with “S1, S2, S3 and S4         pocket” is meant the amino acids of a protease which interact         with the amino acids of an (oligo)peptide acyl donor. The         C-terminal amino acid (1^(st) amino acid; P1) of the acyl donor         (oligo)peptide interacts with the amino acids in the Si pocket         of the protease. The penultimate amino acid (2^(nd) amino acid;         P2) of the acyl donor (oligo)peptide interacts with the amino         acids in the S2 pocket of the protease, the third amino acid         (P3) with the S3 and the fourth amino acid (P4) with the S4         pocket. The S1-S4 binding pockets of a protease are defined by         several amino acids which can be distant in the primary         structure of the protease, but are close in the three         dimensional space. For the purpose of this invention, with S1′         and S2′ pockets are meant the amino acids of a protease which         interact with the N-terminal amino acids of an (oligo)peptide         nucleophile. The N-terminal amino acid of the (oligo)peptide         nucleophile interacts with the amino acids in the S1′ pocket of         the protease. The N-terminal penultimate amino acid of the         (oligo)peptide nucleophile interacts with the amino acids in the         S2′ pocket of the protease. The S1′ and S2′ binding pockets of a         protease are defined by several amino acids which can be distant         in the primary structure of the protease, but are close in the         three dimensional space.     -   For the purpose of this invention, with “denaturating agent” is         meant an additive which potentially can destroy the three         dimensional structure of a protease, and thus, can potentially         inactivate the protease.     -   In the context of the invention with “amino acid side-chain” is         meant any proteinogenic or non-proteinogenic amino acid         side-chain.     -   Proteinogenic amino acids are the amino acids that are encoded         by the genetic code. Proteinogenic amino acids include: alanine         (Ala), valine (Val), leucine (Leu), isoleucine (Ile), serine         (Ser), threonine (Thr), methionine (Met), cysteine (Cys),         asparagine (Asn), glutamine (Gln), tyrosine (Tyr), tryptophan         (Trp), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu),         histidine (His), lysine (Lys), arginine (Arg), proline (Pro) and         phenylalanine (Phe). Selenocysteine (Sec, U) is an amino acid,         of which the structure corresponds to cysteine, with the proviso         that it contains a selenium instead of a sulphur atom.     -   Non-proteinogenic amino acids may in particular be selected         amongst D-amino acids, L- or D-phenylglycine, DOPA         (3,4-dihydroxy-L-phenylalanine), beta-amino acids,         4-fluoro-phenylalanine, or C^(α)-alkylated amino acids.     -   The term “mutated” or “mutation” as used herein regarding         proteins or polypeptides—in particular enzymes—means that at         least one amino acid in the wild-type or naturally occurring         protein or polypeptide sequence has been replaced with a         different amino acid, inserted into, appended to, or deleted         from the sequence via mutagenesis of nucleic acids encoding         these amino acids. Mutagenesis is a well-known method in the         art, and includes, for example, site-directed mutagenesis by         means of PCR or via oligonucleotide-mediated mutagenesis as         described in Sambrook et al., Molecular Cloning-A Laboratory         Manual, 2nd ed., Vol. 1-3 (1989). The term “mutated” or         “mutation” as used herein regarding genes means that at least         one nucleotide in the nucleic acid sequence of that gene or a         regulatory sequence thereof, has been replaced with a different         nucleotide, has been inserted into, has been appended to, or has         been deleted from the sequence via mutagenesis, resulting in the         transcription of a protein sequence with a qualitatively of         quantitatively altered function or resulting in the knock-out of         that gene.     -   In the present specification, a shorthand for denoting amino         acid substitutions employs the single letter amino acid code of         the amino acid that is substituted, followed by the number         designating where in the protein amino acid sequence the         substitution is made. This number is the amino acid position of         the wild-type amino acid sequence (generally subtilisin BPN'         unless specified otherwise). Thus for the mutated amino acid         sequence it is the amino acid position corresponding to the         position with that number in the wild type enzyme. Due to one or         more other mutations at a lower position (additions, insertions,         deletions, etc.) the actual position does not need to be the         same. The skilled person will be able to determine the         corresponding positions using a generally known alignment         technique, such as NEEDLE. The number is followed by the single         letter code of the amino acid that replaces the wild-type amino         acid therein. For example, G166S denotes the substitution of         glycine at the position corresponding to position 166 to serine.         X is used to indicate any other proteinogenic amino acid than         the amino acid to be substituted. For example, G166X denotes the         substitution of glycine 166 to any other proteinogenic amino         acid.

When referring to a compound of which stereoisomers exist, the compound may be any of such stereoisomers or a mixture thereof. Thus, when referred to, e.g., an amino acid of which enantiomers exist, the amino acid may be the L-enantiomer, the D-enantiomer or a mixture thereof. In case a natural stereoisomer exists, the compound is preferably a natural stereoisomer.

The term ‘pH’ is used herein for the apparent pH, i.e. the pH as measured with a standard, calibrated pH electrode.

When an enzyme is mentioned with reference to an enzyme class (EC) between brackets, the enzyme class is a class wherein the enzyme is classified or may be classified, on the basis of the Enzyme Nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which nomenclature may be found at http://www.chem.qmul.ac.uk/iubmb/enzyme/. Other suitable enzymes that have not (yet) been classified in a specified class but may be classified as such, are meant to be included.

Homologues typically have an intended function in common with the polynucleotide respectively polypeptide (enzyme) of which it is a homologue, such as encoding the same peptide respectively being capable of catalyzing the same reaction. The term homologue is also meant to include nucleic acid sequences (polynucleotide sequences) which differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

Amino acid or nucleotide sequences are said to be homologous when exhibiting a certain level of similarity. Two sequences being homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by “percent identity” or “percent similarity”, which is high or low respectively.

The terms “homology”, “percent homology”, “percent identity” or “percent similarity” are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the complete sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment is carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids or amino acids. The percentage identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Other matrices can be specified. The optional parameters used for alignment of amino acid sequences are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

The homology or identity between the two aligned sequences is calculated as follows: the number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as “longest-identity”. For purposes of the invention the level of identity (homology) between two sequences (amino acid or nucleotide) is calculated according to the definition of “longest-identity” as can be carried out by using the program NEEDLE.

The polypeptide sequences representing an enzyme of the present invention, can further be used as a “query sequence” to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults:

Cost to open gap: default=5 for nucleotides/11 for proteins

Cost to extend gap: default=2 for nucleotides/1 for proteins

Penalty for nucleotide mismatch: default=−3

Reward for nucleotide match: default=1

Expect value: default=10

Wordsize: default=11 for nucleotides/28 for megablast/3 for proteins

Furthermore the degree of local identity (homology) between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly the program calculates the identity only for these matching segments. Therefore the identity calculated in this way is referred to as local identity.

The term “homologue” is used herein in particular for polypeptides (enzymes) having a sequence identity of at least 50%, preferably at least 60%, more preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% with the polypeptide (enzyme) with which the homologue peptide is compared. Evidently, the sequence identity will be less than 100%. The percentage of sequence identity will depend on the number of mutations and the length of the polypeptide with which the homologue is prepared. In particular, for a subtilisin BPN′ variant, the number of mutations for the enzymes in the present invention will typically be at least 11, of which at least nine mutations are deletions and at least two mutations are replacements for another amino acid. In ‘longest identity’ alignment the deletions are not taken into account. This means that the sequence identity of an enzyme of the invention compared to subtilisin BPN' generally is 99.25% (two replacements in a polypeptide with 266 amino acids) or less. Preferably, the sequence identity of an enzyme of the invention compared to SEQUENCE ID NO 2, is 98% or less, more preferably 96% or less, in particular 94% or less, more in particular 92% or less, or 90% or less.

“Expression” refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.

As used herein, “heterologous” in reference to a nucleic acid or protein is a nucleic acid or protein that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.

The term “heterologous expression” refers to the expression of heterologous nucleic acids in a host cell. The expression of heterologous proteins in suitable host cell systems are well known to those of skill in the art. The skilled person will be able to provide suitable host cells for producing an enzyme of the invention from various organisms without undue burden based upon common general knowledge and the information disclosed herein. .

As used herein “promoter” is a DNA sequence that directs the transcription of a (structural) gene. Typically, a promoter is located in the 5′ region of a gene, proximal to the transcriptional start site of a (structural) gene. Promoter sequences may be constitutive, inducible or repressible. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent.

The term “vector” as used herein, includes reference to an autosomal expression vector and to an integration vector used for integration into the chromosome.

The term “expression vector” refers to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide (enzyme) of interest under the control of (i.e. operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

“Plasmid” refers to autonomously replicating extrachromosomal DNA which is not integrated into a microorganism's genome and is usually circular in nature.

An “integration vector” refers to a DNA molecule, linear or circular, that can be incorporated in a microorganism's genome and provides for stable inheritance of a gene encoding a polypeptide of interest. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of (i.e. operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the target cell, but which has a replicon which is nonfunctional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment.

As used herein, the term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to another control sequence and/or to a coding sequence is ligated in such a way that transcription and/or expression of the coding sequence is achieved under conditions compatible with the control sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

By “host cell” is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as bacterial cells, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells.

“Transformation” and “transforming”, as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

-   -   The term ‘C-terminal protection’ is used herein to indicate that         a C-terminal carboxylic group of an (oligo)peptide is provided         with a protective group, generally substantially protecting the         carboxylic group from being coupled to an N-terminal amine group         of another (oligo)peptide or of the same (oligo)peptide         molecule. The C-terminal protective group may be a t-alkyl ester         group for instance a t-butyl ester group, which is a commonly         used protective group. The C-terminal protective group may also         be a C-terminal carboxy-amide. A primary carboxy-amide is a         commonly used protective group.     -   The term ‘N-terminal protection’ is used herein to indicate that         an N-terminal amine group of an (oligo)peptide is provided with         a protective group, generally at least substantially protecting         the N-terminal amine group from being coupled to a C-terminal         carboxylic group of another (oligo)peptide or of the same         (oligo)peptide molecule.     -   The (oligo)peptide C-terminal ester or thioester typically is an         activated (thio)ester, i.e. it contains a carboxy ester or         carboxy thioester group that can take part in the enzymatic         coupling reaction. In principle, any (substituted or         unsubstituted) alkyl or (substituted or unsubstituted) aryl         (thio)ester can be used. Typical examples of (thio)esters which         can take part in the enzymatic coupling reaction are methyl-,         ethyl, propyl-, isopropyl-, phenyl-, benzyl-,         2,2,2-trichloroethyl-, 2,2,2-trifluoroethyl-, cyanomethyl- and         carboxyamidomethyl-(thio)esters.

Particularly good results have been obtained with carboxyamidomethyl-type esters represented by the formula peptide-(C═O)—O—CX₁X₂—C(═O)N—R₁R₂. Herein, each X₁ and X₂ independently represents a hydrogen atom or an alkyl group. Good results have been achieved when both X₁ and X₂ are a hydrogen atom (peptide-(C═O)—O—CH₂—C(═O)N—R₁R₂). Herein R₁ represents a hydrogen atom or an alkyl group and R₂ represents a hydrogen atom or an alkyl group or an amino acid or a peptide residue with a C-terminal carboxyamide or carboxylic acid functionality, optionally protected on the side-chain functionality of the amino acid or on one or more of the side-chain functionalities of the amino acids. Herein, each alkyl group may independently represent a (substituted or unsubstituted) C1-C7 alkyl group, preferably a (substituted or unsubstituted) linear C1-C6 alkyl group, more preferably a (substituted or unsubstituted) linear C1-C3 alkyl group, and most preferably a methyl group. Good results have in particular been achieved in a method of the invention wherein both R₁ and R₂ represent a hydrogen atom or wherein R₁ represents a hydrogen atom and R₂ represents an amino acid or peptide residue with a C-terminal carboxyamide or carboxylic acid functionality, optionally protected on the side-chain functionality of the amino acid or on one or more of the side-chain functionalities of the amino acids. Particularly good results have been achieved when using the Cam-ester, when X₁, X₂, R₁ and R₂ are a hydrogen atom.

-   -   The (oligo)peptide C-terminal (thio)ester can be N-terminally         unprotected or N-terminally protected. In an embodiment, one or         more side-chain functionalities (in particular carboxyl groups,         amine groups), e.g. all side-chain functionalities, are provided         with a protecting group; in another embodiment all the         side-chain functionalities are unprotected. In a preferred         embodiment, only the side-chain functionalities of the amino         acids at the P4 and P1 position of the (oligo)peptide acyl donor         and at the P1′ or P2′ position of the (oligo)peptide nucleophile         (in particular hydroxy groups, carboxyl groups or amine groups)         are provided with a protecting group. Suitable protecting groups         are known to the person skilled in the art. Carboxylic acid         groups can for instance be protected with a cyclohexyl, benzyl         or allyl group; amine functionalities can for instance be         protected with an allyloxycarbonyl group or a trifluoroacetyl         group.     -   The activated C-terminal (thio)ester group of the (oligo)peptide         C-terminal (thio)ester can be synthesized using solid phase         synthesis in high yield and purity without racemization. An         additional advantage of the use of (thio)esters wherein R₁         represents a hydrogen atom and R₂ represents an amino acid or         peptide residue with a C-terminal carboxylic acid functionality,         optionally protected on the side-chain functionality of the         amino acid or on one or more of the side-chain functionalities         of the amino acids is, that their activated C-terminal ester or         thioester group can be synthesized using the cheap and         industrially available 2-chlorotritylchloride resin.     -   The activated C-terminal (thio)ester group of the (oligo)peptide         C-terminal (thio)ester can also be synthesized by fermentation         using a microorganism. A reliable method to obtain         (oligo)peptide (thio)esters using fermentation is via so-called         intein expression (see for instance E. K. Lee, Journal of         Chemical Technology and Biotechnology, 2010, 9, 11-18).         Different intein expression systems kits are commercially         available (for instance the IMPACT™ kit). Other methods for the         fermentative production of (oligo)peptide (thio)esters are known         in the art.     -   The C-terminal amino acid of the (oligo)peptide C-terminal         (thio)ester and the other amino acids of the (oligo)peptide         C-terminal (thio)ester may in principle be any amino acid,         proteinogenic or non-proteinogenic. If the amino acid sequence         of the C-terminal part of the (oligo)peptide C-terminal         (thio)ester is poorly recognized by or inaccessible to the         coupling enzyme due to the amino acid preference of the coupling         enzyme and/or due to the secondary or tertiary structure of the         (oligo)peptide, the primary structure (amino acid sequence) may         be elongated at the C-terminus. Essentially the C-terminus of         the (oligo)peptide C-terminal (thio)ester is elongated with a         number of amino acids to ensure good recognition by the enzyme         and accessibility into the enzyme for the enzymatic coupling         reaction. The skilled person will know how to elongate the         (oligo)peptide C-terminal (thio)ester on the basis of the         information disclosed herein and common general knowledge.         Usually the number of amino acids for elongation is in the range         of 1-10, although in principle it can be higher. Good results         have been obtained by elongation of the (oligo)peptide         C-terminal (thio)ester with 4 amino acid residues, e.g.         -Phe-Ser-Lys-Leu-(thio)ester.     -   In particular the (optionally N-terminal protected)         (oligo)peptide C-terminal (thio)ester may be represented by a         compound of Formula I.

-   -   Herein Q represents an OR or SR moiety. R may represent a         (substituted or unsubstituted) alkyl or a (substituted or         unsubstituted) aryl group.     -   Herein P¹ stands for a hydrogen or an N-terminal protecting         group. Suitable N-terminal protecting groups are those         N-protecting groups which can be used for the synthesis of         (oligo)peptides. Such groups are known to the person skilled in         the art. Examples of suitable N-protecting groups include         carbamate or acyl type protecting groups, for instance ‘Cbz’         (benzyloxycarbonyl), ‘Boc’ (tert-butyloxycarbonyl), ‘For’         (formyl), ‘Fmoc’ (9-fluorenylmethoxycarbonyl), ‘PhAc’         (phenacetyl) and ‘Ac’ (acetyl). The groups For, PhAc and Ac may         be introduced and cleaved enzymatically using the enzymes         Peptide Deformylase, PenG acylase or Acylase, respectively.         Chemical cleavage methods are generally known in the art.     -   Herein, n is an integer of at least 2. n May in particular be at         least 3, at least 4, at least 5, at least 6, at least 7 at least         8, at least 9 or at least 10. n May in particular be 100 or         less, 75 or less, 50 or less, 25 or less, 20 or less 15 or less,         e.g. 10 or less.     -   Herein, each R^(A) and each R^(B) independently represent a         hydrogen atom or an organic moiety, preferably an amino acid         side-chain. Thus, it is not required that R^(A) is the same in         all n amino acid units. Similarly, it is not required that R^(B)         is the same in all n amino acid units. Optionally, one or more         of the side-chain functionalities may contain a protecting         group.     -   The amino acid units of the (oligo)peptide nucleophile may in         principle be selected from any amino acid, proteinogenic or         non-proteinogenic.     -   In particular, the (oligo)peptide nucleophile may be represented         by a compound of Formula II.

-   -   Herein, n, R^(A) and R^(B) are as defined above.     -   Herein P² represents an amine moiety or an OR moiety.     -   In case P² represents an amine moiety, the amine moiety may be         represented by the formula NR₃R₄, in which R₃ and R₄ may each         individually represent any (substituted or unsubstituted) alkyl         or (substituted or unsubstituted) aryl group. In particular, one         out of R₃ and R₄ is a hydrogen atom and the other a (substituted         or unsubstituted) alkyl group. Good results have particularly         been obtained with R₃ and R₄ both being a hydrogen atom.     -   In case P² represents an OR moiety, R may represent a C-terminal         protective group or a cation, for instance a monovalent cation,         such as a tri- or tetrasubstituted ammonium ion or an alkaline         metal cation or an H. In case R is a C-terminal protective group         this may in particular be an optionally substituted alkyl group.         Preferably it is a t-alkyl group, although in principle it also         may be any other protective ester as known to a man skilled in         the art. The t-alkyl may in principle be any protective tertiary         alkyl group. Preferably the t-alkyl is selected from the group         of t-butyl (2-methyl-2-propyl), t-pentyl (2-methyl-2-butyl) and         t-hexyl (2,3-dimethyl-2-butyl).     -   In an embodiment, the (oligo)peptide nucleophile is C-terminal         protected. In another embodiment it is not C-terminal protected.

The (oligo)peptide nucleophile may be synthesized using methods known in the art, such as solid-phase synthesis, solution phase synthesis or by fermentation using a microorganism. The N-terminal amino acid of the (oligo)peptide nucleophile and the other amino acids of the (oligo)peptide nucleophile may in principle be any amino acid, proteinogenic or non-proteinogenic. If the amino acid sequence of the N-terminal part of the (oligo)peptide nucleophile is poorly recognized by or inaccessible to the coupling enzyme due to the amino acid preference of the coupling enzyme or due to the secondary or tertiary structure of the (oligo)peptide nucleophile, the primary structure (amino acid sequence) may be elongated at the N-terminus. Essentially the N-terminus of the (oligo)peptide nucleophile is elongated with a number of amino acids to ensure good recognition by and accessibility to the coupling enzyme for the enzymatic coupling reaction. The skilled person will know how to elongate the (oligo)peptide nucleophile on the basis of the information disclosed herein and common general knowledge. Usually the number of amino acids for elongation is in the range of 1-10, although in principle it can be higher. Good result have been obtained by elongation of the (oligo)peptide nucleophile with 3 amino acid residues, e.g. H-Ser-Tyr-Arg.

The invention provides an enzyme having catalytic activity with respect to the formation of a peptide bond (condensation activity), whereby it has catalytic activity in the synthesis of an (oligo)peptide with a high S/H ratio. In particular, the enzyme has ligase activity or cyclase activity, i.e. catalytic activity in the cyclization of an (oligo)peptide by catalyzing the formation of a peptide bond by coupling the C-terminus and the N-terminus of an (oligo)peptide.

In particular, the invention provides an isolated enzyme (isolated from the organism wherein it has been expressed (typically a recombinant organism), if it has been produced in an organism or from the reaction medium in which it has been synthesized.

In particular, an enzyme of the invention is considered isolated for the purpose of the invention if it has been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

An enzyme of the present invention can be provided in at least substantially pure form (e.g. more than 75 wt. %, more than 80 wt. %) or in a mixture with one or more other components, e.g. in the form of a stock solution, in particular in an aqueous buffer solution.

This enzyme is typically a subtilisin BPN′ variant or homologue thereof. The present disclosure provides various examples of enzymes of the invention, which are in particular considered subtilisin BPN′ variants. As already described above, an enzyme of the invention should comprise at least:

-   -   a deletion of the amino acids corresponding to L75, N76, N77,         S78, I79, G80, V81, L82 and G83 of subtilisin BPN′ (475-83; thus         in general a deletion of a corresponding Ca²⁺ binding site)     -   a cysteine or selenocysteine at a position corresponding to         position 221 in subtilisin BPN′     -   preferably an amino acid different from proline at position         corresponding to position 225 in subtilisin BPN′.

It has surprisingly been found that a mutant having both the deletion corresponding to Δ75-83 of subtilisin BPN′ and the mutation to a cysteine corresponding to position 221 in subtilisin BPN′ has sufficient stability and an S/H ratio of more than 1, which is an improved S/H ratio compared to, e.g. subtiligase. The position corresponding to S221 in a subtilisin is considered to be important for stability and activity of the enzyme, and of alcalase it has been reported that a single mutation corresponding to S221C results in a virtually inactive enzyme. In this respect, good results have been achieved with the mutation into cysteine at a position corresponding to position 221.

An enzyme of the invention may have further mutations compared to subtilisin BPN′, provided that it has enzymatic fragment condensation or cyclisation activity in the preparation of an (oligo)peptide, in particular one or more further mutations as described elsewhere herein.

Alternatives to subtilisin BPN′, as template enzymes from which an enzyme according to the invention, in particular a homologue of a subtilisin BPN′ variant of the invention, can be derived by mutagenesis are other subtilisins, in particular subtilisins having at least 50% homology with subtilisin BPN′.

Sequences of suitable subtilisins can be retrieved from the UNIPROT sequence database (http://www.uniprot.org/), as available on 11 Aug. 2014, by BLASTing the database with subtilisin BPN′ (SEQ ID 2) as a query. However sequence retrieval is not limited to UNIPROT nor to the date. The skilled person in the art knows how to query alternative sequence depositories or to collect additional homologue sequences by sequencing (see for example Zooming in on metagenomics: molecular microdiversity of Subtilisin Carlsberg in soil., Gabor E, Niehaus F, Aehle W, Eck J. J Mol Biol. 2012 Apr. 20; 418(1-2):16-20). In particular, the invention further relates to variants, having at least said deletions of the amino acids corresponding to L75 till and including G83 of subtilisin BPN′, cysteine at a position corresponding to position 221 in subtilisin BPN′ and alanine or another mutation at position corresponding to position 225 in subtilisin BPN′ (such as a mutation corresponding to P225N, 225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, P225Q of SEQUENCE ID NO: 2) of any of the subtilisins mentioned in FIG. 14, of which the full sequence is as available from said UNIPROT sequence data base and of which the alignments around positions 75-83 are shown.

Preferably, the subtilisin BPN′ variant or homologue of the invention comprises a mutation at the position corresponding to P225. For an improvement in S/H ratio, the mutation is usually a mutation corresponding to P225 selected from the group of P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, P225Q, P225F and P225E. For an improvement of the S/H ratio compared to, e.g., subtiligase a mutation is preferred corresponding to P225 selected from the group of P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H and P225Q. Of these, particularly good results have been achieved with said mutation into one of the amino acids of the group commonly referred to as ‘Asx’, i.e. asparagine (Asn/N) and aspartic acid (Asp/D), i.e. the mutation corresponding to P225N or P225D. Further, particularly good results have been achieved with the mutation corresponding to P225S. Further, particularly good results have been achieved with the mutation corresponding to P225C.

Further, good results have been achieved with the mutation corresponding to P225G. Further, good results have been achieved with the mutation corresponding to P225A. Further, good results have been achieved with the mutation corresponding to P225T. Further, good results have been achieved with the mutation at the position corresponding to P225 into a branched amino acid, i.e. valine (V), isoleucine (I) or Leucine (L).

Preferably, the subtilisin BPN' variant or homologue of the invention comprises one or more mutations at an amino acid position corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218S, T254 and Q271 of SEQUENCE ID NO 2. The inventors found that one or more of the following mutations are advantageous in the subtilisin BPN′ variant of the invention: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A and Q271E. In particular for an improved activity, an improved stability or an improved S/H ratio it is preferred that a plurality of said mutations are present in an enzyme of the invention, such as at least two, at least three, more preferably four or more, more preferably five or more, more preferably six or more, more preferably at least eight, more preferably at least 12 of the mutations selected from the group of Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A and Q271E. The inventors consider that in particular the presence of one or more of the mutations N218S, S3C-Q206C, G169A, T254A, A73L, M50F and Q2K are advantageous with respect to improving enzyme stability. Further, the inventors consider that in particular the presence of one or more of the mutations I31L, E156S, G166S, G169A, is advantageous with respect to improving activity and/or S/H ratio.

Further, a subtilisin BPN′ variant or homologue according to the invention comprising a plurality of mutations at an amino acid position corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218S, T254 and Q271 of SEQUENCE ID NO 2 is easier to produce and purify than subtiligase.

In a preferred embodiment, the enzyme, comprises a mutation at the amino acid position corresponding to N218, in particular N218S.

In a preferred embodiment, the enzyme, comprises a mutation at the amino acid position corresponding to M50, in particular M50F.

In a preferred embodiment, the enzyme comprises a mutation at the amino acid position corresponding to Q2, in particular Q2K.

In a preferred embodiment, the enzyme comprises a mutation at the amino acid position corresponding to A73, in particular A73L.

In a preferred embodiment, the enzyme comprises a mutation at the amino acid position corresponding to P5, in particular P5S.

In a preferred embodiment, the enzyme comprises a mutation at the amino acid position corresponding to G166, in particular G166S.

In a preferred embodiment, the enzyme comprises a mutation at the amino acid positions corresponding to S3 and Q206, in particular S3C-Q206C

For an improved S/H ratio, it is particularly preferred that the enzyme comprises a mutation at each of the positions corresponding to Q2, P5, M50, A73 and N218, more in particular at each of the positions corresponding to Q2, P5, M50, A73, G166 and N218.

In particular, good results have been achieved with a subtilisin BPN′ variant comprising each of the mutations corresponding to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A and Q271E.

Further, it has surprisingly been found that the S/H ratio is improved in general or for certain substrates, by altering the S1′ pocket or the S4 pocket by site-specific mutations in one or more of the amino acid positions of said pockets. It is in particular surprising that a site-specific mutation in a pocket, in particular the P1′ pocket has an effect in another pocket, in particular the P2′ pocket. The inventors realized that this is also advantageous for broadening the range of substrates that can advantageously be used in a method for synthesizing a peptide according to the invention. Thus this broadens the substrate scope for which an enzyme of the invention offers a high S/H ratio.

The S1′ pocket is mainly formed by the amino acids M222 and Y217 (Strausberg L. et al. Biochemistry, 2005, 44, 3272; Estell D. A. et al. J. Biol. Chem., 1985, 260, 6518). The three dimensional structure of the S1′ binding pocket may also be altered by more distant amino acids, for instance N62, G100, S125, L126, G127, P129, N155 and N218. Substitution of one or more of these amino acids may significantly alter and improve the S/H ratio of the subtilisin BPN′ variant or homologue, at least for a number of peptide sequences. In an advantageous embodiment, the substitution at an amino acid position corresponding to M222 or Y217 increases the activity, S/H ratio or substrate scope for which the enzyme shows a (highly) improved S/H ratio.

Preferably, a mutation is present at the position corresponding to M222 is M222G, M222P, M222N, M222E, M222Q or M222A. In a particularly preferred embodiment, said mutation corresponds to M222P or M222G.

Preferably, a mutation at the position corresponding to Y217 is Y217L, Y217N, Y217E, Y217G, Y217S, Y217F or Y217H.

Particularly good results have been obtained with a variant having a mutation selected from the group of M222G, M222P and Y217L in that the S/H ratio and/or the activity of the resulting subtilisin BPN′ variant significantly increases, at least for a number of peptide sequences.

The S4 binding pocket is mainly formed by the amino acids Y104, I107, L126, S101, G102, G127, and G128, but the three-dimensional structure of the S4 binding pocket is also determined by more distant amino acids such as L135 and P168 (Ruan et al. Biochemistry, 2008, 47, 6628; Rheinnecker et al. Biochemistry, 1994, 33, 221).

Preferably, the enzyme comprises a mutation at one, two or each of the positions corresponding to Y104, I107 and L135. Particularly good results have been obtained with a subtilisin BPN′ variant having a mutation selected from the group of Y104F, Y1045, I107V, I107A, L135N, L135S, L135D and L135A. Substitution of these amino acids can significantly alter and improve the S/H ratio and/or the activity of the enzyme, at least for certain substrates.

In particular, good results with respect to P4 substrate scope, enzyme activity and S/H ratio have been obtained with a subtilisin BPN′ variant having a substitution in the amino acid corresponding to I107 (I107V) and a substitution in L135 (L135S or L135N).

In a preferred embodiment, the enzyme of the invention has one or more substitutions in the S1′ binding pocket and one or more substitutions in the S4 binding pocket, in particular two or more substitutions in the S1′ binding pocket and two or more substitutions in the S4 binding pocket.

A substitution of both the amino acids corresponding to M222 and I107 has been found advantageous for providing an enzyme with improved activity and S/H ratio compared to a variant of the invention having only one of said mutations. Either mutation alone was also found beneficial for the enzyme activity and S/H ratio. In particular, good results in such embodiment have been achieved by mutations I107V and M222G. Examples of other combinations of mutations of specific interest are variants with mutations L135N+M222G and variants with mutations I107V+M222P. Further, such a combination of mutations at the positions corresponding to I107 and M222 offers improvement with respect to substrate scope for both the P4 and the P1′ pocket.

In a preferred embodiment, the subtilisin BPN′ variant or homologue thereof according to the invention, has a substitution in the S1′ binding pocket at the position corresponding to M222 and at the position corresponding to Y217. The M222 mutation in this embodiment preferably is either M222G or M222P. The Y217 mutation preferably is one selected from the group of Y217F, Y217H and Y217G. Such enzymes of the invention have been found to have a broad substrate scope and a good S/H ratio. Particularly good results have been achieved with a subtilisin BPN′ variant or homologue thereof comprising the mutations M222P and Y217H; the mutations M222P and Y217G; the mutations M222G and Y217F; or the mutations M222G and Y217G. Of these, a subtilisin BPN′ variant or homologue thereof comprising the mutations M222G and Y217F gave particularly good results with respect to substrate broadness and S/H ratio.

Good results have been achieved with a subtilisin BPN′ variant or homologue thereof according to the invention having a substitution in the S1′ binding pocket at the position corresponding to M222 and at the position corresponding to Y217 that is free of mutations in the S4 binding pocket. However, in an alternative embodiment, with which also good results have been achieved, it additionally has one or more mutations in the S4 binding pocket. In a specific embodiment, this subtilisin BPN′ variant or homologue thereof has a substitution in two or more positions of the S4 binding pocket corresponding to Y104, I107, L126, L135, S101, G102, G127, and G128. The mutations in the S4 binding pocket may in particular include I107V and/or either L135N or L135S.

Preferred enzymes according to the invention are in particular the subtilisin BPN′ variant or homologues comprising any one of the sequences represented by SEQUENCE ID NO 3, 4 or 5 or homologues thereof. SEQUENCE ID NO 3 shows the preferred mutation corresponding to S221C, although in another embodiment this can be selenocysteine. The X at the position corresponding to P225 can be P, or a different amino acid preferably one of the preferred mutations identified elsewhere herein (N/D/S/C/G/A/T/V/I/LH/Q). SEQUENCE ID NO 4 shows preferred mutation sites compared to SEQUENCE ID NO 3. In SEQUENCE ID NO 4, each X independently represents any proteinogenic amino acid. In particular, any X can be the amino acid present in the wild type subtilisin BPN′ at the position of that X or a mutation as described elsewhere in the present disclosure. Preferably, one or more X's represent a mutation, as described elsewhere herein.

In the method of the invention the enzymatic coupling reactions and cyclisations are performed in a fluid comprising water. Preferably the reaction is performed in a buffered fluid. The water content usually is 10-100 vol %, based on total liquids, preferably 20 vol. % or more, preferably 40 vol. % or more, in particular 50 vol. % or more in particular 60 vol. % or more.

In principle, any buffer is suitable. Good buffers are known to a person skilled in the art. See for instance David Sheehan in Physical Biochemistry, 2^(nd) Ed. Wiley-VCH Verlag GmbH, Weinheim 2009; http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-calculator.html.

The pH of the buffer for an (oligo)peptide fragment condensation may be at least 5, in particular at least 6, preferably at least 7. A desired maximum pH is usually less than 11, in particular less than 10, even more preferably less than 9. Usually the optimal pH for the enzymatic reactions is between 7 and 9. For cyclisation reactions the optimal pH can be different. The pH for the cyclisation reaction may be at least 3, in particular at least 4, preferably at least 5. A desired maximum pH is usually less than 11, in particular less than 10, preferably less than 9. Usually the optimal pH for the enzymatic cyclisation reactions is between 5 and 9.

Due to the high S/H ratio, a large excess of the (oligo)peptide C-terminal ester or thioester or of the (oligo)peptide nucleophile is generally not needed to reach a high yield in the condensation reaction. Usually the ratio of (a) the (oligo)peptide C-terminal ester or thioester to (b) the (oligo)peptide nucleophile is between 1:5 and 5:1, preferably in the range of 1:3 to 3:1, more preferably in the range of 1.0:2.5 to 2.5:1.0, in particular in the range of 1:2 to 2:1, more in particular in the range of 1:1.5 to 1.5:1. An about stoichiometric ratio has been found particularly effective.

In the method of the invention, it may be advantageous to add additives to the fluid wherein the reaction is carried out to improve the solubility of the (oligo)peptide fragments or to improve the reaction yield. Such additives may be a salt or an organic molecule, for instance guanidinium hydrochloride, urea, sodium dodecasulphate or Tween.

The reaction may be carried out in a fully aqueous liquid or in a mixture of water and a water mixable co-solvent such as N,N-dimethylformamide (DMF), N-methyl-pyrrolidinone (NMP), N,N-dimethylacetamide (DMA), dimethyl sulphoxide (DMSO), acetonitrile, an ether, such as tetrahydrofuran (THF), 2-methyl-tetrahydrofuran (Me-THF) or 1,2-dimethoxyethane, or a (halogenated) alcohol, such as methanol, ethanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoroisopropanol, or a mixture of these organic solvents. Depending on the stability of the subtilisin BPN′ variant and the solubility of the (oligo)peptide substrates, the amount of co-solvent is preferably below 70 vol %, more preferably below 60 vol %, even more preferably below 50 vol %, and most preferably below 40%.

In principle the temperature during the enzymatic fragment condensations or cyclisations is not critical, as long as a temperature is chosen at which the subtilisin BPN′ variant used show sufficient activity and stability. Such a temperature is usually known for the subtilisin BPN′ variant to be used or can be routinely determined, making use of a known substrate for the subtilisin BPN′ variant under known reaction conditions. Generally, the temperature may be at least −10° C., in particular at least 0° C. or at least 10° C. Generally, the temperature may be 70° C. or less, in particular 60° C. or less or 50° C. or less. Optimal temperature conditions can easily be identified for a specific subtilisin BPN′ variant for a specific enzymatic fragment condensation or cyclisation by a person skilled in the art through routine experimentation based on common general knowledge and the information disclosed herein. In general, the temperature advantageously is in the range of 20-50° C.

The subtilisin BPN′ variants of the present invention are generally produced by recombinant methods, in particular by expression of a subtilisin BPN′ DNA which has been mutated such that upon expression it results in a subtilisin BPN′ variant of the invention which is enzymatically active.

Expression of the DNA of the subtilisin BPN′ variants and homologues thereof of the present invention is provided using available vectors and regulatory sequences. The actual selection depends in large part upon the particular host cells which are utilized for expression. For example, if the subtilisin BPN′ mutant DNA is expressed in Bacillus, a Bacillus promoter is generally utilized as well as a Bacillus derived vector.

In order to produce and secrete the enzyme of the invention from a host cell into the medium, a gene may be used which encodes a precursor polypeptide (enzyme) containing a signal sequence and a pre-pro sequence preceding the mature enzyme. In subtilisin BPN′, the additional N-terminal sequence comprises 107 amino acids. Upon secretion first the signal sequence can be removed and after secretion the pre-pro sequence can be removed resulting in the fully active enzyme (James A. Wells, Nucleic Acids Research, Volume 11 Number 22 1983). In case of native subtilisin BPN′ the mature enzyme comprises 275 amino acids. Conveniently to describe the position of individual amino acids in the polypeptide chain of subtilisin BPN′ and its homologues the so called subtilisin BPN′ numbering is used which runs from the N-terminus (amino acid 1) tot the C-terminus (amino acid 275). Corresponding positions in homologous enzymes can be determined by aligning said homologous sequences with the sequence of subtilisin BPN′.

As is known to the person skilled in the art, it is possible that the N- and/or C-termini of the mature polypeptide numbered 1-275 within SEQ ID NO: 5 or of the mature enzyme in the amino acid sequence according to SEQ ID NO: 2, 3 or 4 (as set out in amino acids 1 to 275) maybe heterogeneous, due to variations in processing during maturation. In particular such processing variations might occur upon overexpression of the enzyme. In addition, exo-protease activity might give rise to heterogeneity. The extent to which heterogeneity occurs depends also on the host and fermentation protocols that are used. Such C-terminal processing artefacts might lead to shorter polypeptides or longer polypeptides than indicated with the mature wild-type subtilisin BPN' (SEQ ID NO: 2) or with the mature enzymes according to the invention represented by SEQ ID NO: 3 or 4. As a result of such processing variations the N-terminus might also be heterogeneous. Processing variants at the N-terminus could be due to alternative cleavage of the signal sequence by signal peptidases.

The enzyme of the invention may be produced by recombinant technology, based on common general knowledge and the information disclosed herein. For secretion of the translated enzyme into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, an appropriate secretion signal sequence may be fused to the polynucleotide encoding the enzyme of the invention. The signals may be endogenous to the enzyme or they may be heterologous signals.

The enzyme according to the invention may be produced in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids (a so called tag), particularly charged amino acids, may be added to the enzyme, in particular to the C-terminus of the enzyme, to improve stability and persistence in the host cell, during purification or during subsequent handling and storage or to facilitate the purification. Examples of suitable tags are for instance described in a review by M. E. Kimple et al., in ‘Current Protocols in Protein Science 9.9.1-9.9.23, August 2013’. A well known example of a useful tag is the so called His tag, an amino acid sequence having a plurality of histidine units. The inventors found that such a tag could be used successfully in the production and purification of enzymes of the invention. No substantial differences in functional enzyme properties were observed between enzymes with the His tag and enzymes without the His tag.

Further, an enzyme of the invention can be produced as an inclusion body with refolding in an appropriate buffer.

Enzymes of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the enzymes of the present invention may be glycosylated or may be non-glycosylated. In addition, enzymes of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

Polynucleotides of the invention can be incorporated into a vector, including cloning and expression vectors. A vector may be a recombinant replicable vector. The vector may be used to replicate a polynucleotide of the invention in a compatible host cell. The vector may conveniently be subjected to recombinant DNA procedures.

The invention also pertains to methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of an enzyme of the invention occurs. The invention provides a method of making enzymes of the invention by introducing a polynucleotide of the invention into a vector, in an embodiment an expression vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.

The vector may be recovered from the host cell.

A vector according to the invention may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.

Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted. Another type of vector is a viral vector, wherein additional DNA segments can be inserted into the viral genome.

Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., bacterial integration vector without a suitable origin of replication or a non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

The recombinant expression vectors of the invention comprise a polynucleotide of the invention in a form suitable for expression of the polynucleotide in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the polynucleotide sequence to be expressed. The term regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

A vector or expression construct for a given host cell may thus comprise the following elements operably linked to each other in a consecutive order from the 5′-end to 3′-end relative to the coding strand of the sequence encoding an enzyme of the invention: (1) a promoter sequence capable of directing transcription of the nucleotide sequence encoding the enzyme in the given host cell; (2) a ribosome binding site to facilitate the translation of the transcribed RNA (3) optionally, a signal sequence capable of directing secretion of the enzyme from the given host cell into a culture medium; (4) a polynucleotide sequence according to the invention; and preferably also (5) a transcription termination region (terminator) capable of terminating transcription downstream of the nucleotide sequence encoding the enzyme.

Downstream of the nucleotide sequence according to the invention there may be a 3′ untranslated region containing one or more transcription termination sites (e.g. a terminator, herein also referred to as a stop codon). The origin of the terminator is less critical. The terminator can, for example, be native to the DNA sequence encoding the enzyme. However, preferably a bacterial terminator is used in bacterial host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the enzyme is to be expressed). In the transcribed region, a ribosome binding site for translation may be present. The coding portion of the mature transcripts expressed by the constructs will include a start codon is usually AUG (or ATG), but there are also alternative start codons, such as for example GUG (or GTG) and UUG (or TTG), which are used in prokaryotes. Also a stop or translation termination codon is appropriately positioned at the end of the polypeptide to be translated.

Enhanced expression of the polynucleotide of the invention may also be achieved by the selection of homologous and heterologous regulatory regions, e.g. promoter, secretion leader and/or terminator regions, which may serve to increase expression and, if desired, secretion levels of the protein of interest from the expression host and/or to provide for the inducible control of the expression of an enzyme of the invention.

The enzymes according to the invention can be produced in bacterial cells such as E. coli and Bacilli, insect cells (using baculovirus expression vectors), fungal cells, yeast cells or mammalian cells. Suitable host cells are discussed herein and further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) and in “Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems”, 2004, Wiley-Blackwell, Editor (http://eu.wiley.com/WileyCDA/Section/id-302479.html?query=Gerd+Gellissen). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

For most bacteria, filamentous fungi and yeasts, the vector or expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants. In case the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.

In the invention, bacteria, in particular Bacilli, may preferably be used as host cells for the expression of an enzyme of the invention. Suitable inducible promoters useful in such host cells include promoters regulated primarily by an ancillary factor such as a repressor or an activator. The repressors are sequence- specific DNA binding proteins that repress promoter activity. The transcription can be initiated from this promoter in the presence of an inducer that prevents binding of the repressor to the operator of the promoter. Production of secondary sigma factors can be primarily responsible for the transcription from specific promoters. Attenuation and antitermination also regulates transcription.

Strong constitutive promoters are well known and an appropriate one may be selected according to the specific sequence to be controlled in the host cell. A variety of promoters can be used that are capable of directing transcription in the recombinant host cells of the invention. Preferably the promoter sequence is from a highly expressed gene.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via natural competence, conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign polynucleotide (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid mediated transfection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra) and other laboratory manuals.

In order to identify and select cells which harbor a vector, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the polynucleotide of the invention. Preferred selectable markers include, but are not limited to, those which confer resistance to drugs or which complement a defect in the host cell. They also include e.g. versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or genes providing resistance to antibiotics like G418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycin orbenomyl resistance (benA). Alternatively, specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e.g. D-alanine racemase (from Bacillus), URA3 (from S. cerevisiae or analogous genes from other yeasts), pyrG or pyrA (from A. nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC. In an embodiment the selection marker is deleted from the transformed host cell after introduction of the expression construct so as to obtain transformed host cells capable of producing enzymes of the invention which are free of selection marker genes.

Expression of proteins in prokaryotes is often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

Vectors preferred for use in bacteria are for example disclosed in WO-A1-2004/074468, which are hereby enclosed by reference. Other suitable vectors will be readily apparent to the skilled artisan.

Vectors of the invention may be transformed into a suitable host cell as described herein to provide for expression of a polypeptide of the invention. Thus, in a further aspect the invention provides a process for preparing an enzyme according to the invention which comprises cultivating a host cell transformed or transfected with an expression vector encoding the enzyme, and recovering the expressed polypeptide.

A polynucleotide according to the invention encodes, when transformed into a proper host cell an enzyme according to the invention. The invention features cells, e.g., transformed host cells or recombinant host cells comprising a polynucleotide according to the invention or comprising a vector according to the invention. A “transformed host cell” or “recombinant host cell” is a cell into which a polynucleotide according to the invention has been introduced, by means of recombinant DNA techniques.

Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, insect, mammalian and the like.

Suitable host cells include bacteria, including Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus Streptomyces, and Pseudomonas, In an aspect of the vector according to the invention, the host cell is a bacterial cell selected from the group consisting of B. subtilis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.

In a further embodiment of the vector according to the invention the suitable host cell is an Aspergillus, Chrysosporium, Kluyveromyces, Penicillium, Saccharomyces, or Talaromyces species.

Preferably the host cell is a Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Escherichia coli, Aspergillus Niger or Aspergillus oryzae species.

The recombinant host cell according to the invention may comprise the polynucleotide according to the invention or the vector according to the invention. In an embodiment of the recombinant host cell according the invention, the recombinant host cell is capable of expressing or over-expressing the polynucleotide according to the invention or the vector according to the invention.

The method according to the invention for manufacturing the polynucleotide according to the invention or the vector according to the invention comprises the steps of culturing a host cell transformed with said polynucleotide or said vector and isolating said polynucleotide or said vector from said host cell.

Preferred are cells of a Bacillus strain, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,

Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans or Streptomyces murinus; or from a gram negative bacterium, e.g., E. coli or Pseudomonas sp. (Long Liu et a., Appl Microbiol Biotechnol (2013) 97:6113-6127 and Kay Terpe, Appl Microbiol Biotechnol (2006) 72:211-222).

According to another aspect, the host cell is a eukaryotic host cell. In an embodiment the eukaryotic cell is a fungal cell, i.e. a yeast cell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. Preferably the yeast cell is a Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia hpolytica, Pichia pastoris, or a filamentous fungal cell.

Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium and Trichoderma. In an embodiment, filamentous fungal cells are used belonging to a species of an Aspergillus, Chrysosporium, Penicillium, Talaromyces, Fusarium or Trichoderma genus, and preferably a species of Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophthora thermophila, Fusarium oxysporum, Trichoderma reesei or Penicillium chrysogenum.

A host cell can be chosen which modifies and processes the encoded enzyme in a specific, desired fashion after translation. Such post translational modification (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those skilled in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein produced. E.g., in an embodiment a subtilisin BPN′ variant or homologue thereof is initially secreted as a pre-pro-enzyme and the presence of the 77 amino acid pro sequence is important for in vivo production of mature subtilisin but has to be cleaved off to obtain full catalytic activity.

A method of producing an enzyme according to the invention typically comprises cultivating a recombinant host cell e.g. transformed or transfected with an expression vector under conditions to provide for expression of a coding sequence encoding the enzyme and recovering and purifying the produced enzyme from the cell or culture medium. Polynucleotides of the invention can be incorporated into a recombinant replicable vector, e. g . an expression vector or a replication vector. Transcription vectors are used to amplify their insert.

The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene. Simpler vectors called transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their insert. Thus in a further embodiment, the invention provides a method of making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector. The vector may be recovered from the host cell.

Preferably, the enzyme according to the invention is produced as a secreted protein in which case the nucleotide sequence encoding a mature form of the enzyme in the expression construct is operably linked to a nucleotide sequence encoding a signal sequence. Preferably the signal sequence is native (homologous), also referred to herein as “wild type” to the nucleotide sequence encoding the enzyme. Alternatively the signal sequence is foreign (heterologous) to the nucleotide sequence encoding the enzyme, in which case the signal sequence is preferably endogenous to the host cell in which the nucleotide sequence according to the invention is expressed. Examples of suitable signal sequences for bacilli can be found in “van Dijl, J. M. et al. 2001. In: Sonenshein, A. L., Hoch, J. A. and Losick, R., eds. Bacillus subtilis and its closest relatives: from genes to cells. Washington, DC: ASM Press, pp. 337-355” and “Degering C et al., Appl Environ Microbiol. 2010 October; 76(19):6370-6.”

Expression of heterologous proteins in yeast is well known. Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to express proteins in yeast. Vectors, strains, and protocols for expression in, e.g. Saccharomyces and Pichia are generally known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired. More specifically, suitable yeast signal sequences are those from yeast alfa-factor genes. Similarly, a suitable signal sequence for filamentous fungal host cells is e.g. a signal sequence from a filamentous fungal amyloglucosidase (AG) gene, e.g. the A. niger g/aA gene. This may be used in combination with the amyloglucosidase (also called (gluco) amylase) promoter itself, as well as in combination with other promoters. Hybrid signal sequences may also be used with the context of the present invention. Preferred heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (g/aA-both 18 and 24 amino acid versions e.g. from Aspergillus), the [alpha]-factor gene (yeasts e.g. Saccharomyces and Kluyveromyces) or the [alpha]-amylase (amyE, amyQ and amyL) and alkaline protease aprE and neutral protease genes (Bacillus).

A heterologous host cell may also be chosen wherein the enzyme of the invention is produced in a form which is substantially free of enzymatic activities that might interfere with the applications, e.g. free from peptide degrading or modifying enzymes. In particular in the case of producing variants, the host cell should not produce any wild type enzyme. This may be achieved by choosing a host cell which does not normally produce such enzymes or by deliberately removing the corresponding genes by techniques known in the art.

The invention encompasses processes for the production of the enzyme of the invention by means of recombinant expression of a DNA sequence encoding the enzyme of the invention. For this purpose the DNA sequence of the invention can be used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow economic production of the enzyme in a suitable homologous or heterologous host cell. A homologous host cell is a host cell which is of the same species or which is a variant within the same species as the species from which the DNA sequence is obtained. The host cell may over-express the enzyme, and techniques for engineering over-expression are well known. The host may thus have two or more copies of the encoding polynucleotide (and the vector may thus have two or more copies accordingly). Therefore in one embodiment of the invention the recombinant host cell according to the invention is capable of expressing or overexpressing a polynucleotide or vector according to the invention.

Another aspect of the invention is a method for producing an enzyme of the invention comprising (a) culturing a recombinant host cell according to the invention under conditions such that the enzyme of the invention is produced; and (b) optionally recovering the enzyme of the invention from the cell culture medium. For each combination of a promoter and a host cell, culture conditions are available which are conducive to the expression the DNA sequence encoding the enzyme. After reaching the desired cell density or titer of the enzyme the culture is stopped and the enzyme is recovered. The term “culturing” includes maintaining and/or growing a living recombinant host cell of the present invention, in particular the recombinant host cell according to the invention.

In one aspect, a recombinant host cell of the invention is cultured in liquid media. In another aspect, a recombinant host cell is cultured in solid media or semi-solid media. Preferably, the recombinant host cell of the invention is cultured in liquid media comprising nutrients essential or beneficial to the maintenance and/or growth of the recombinant host cell. The recombinant host cells may be cultured in liquid media either continuously or intermittently, by conventional culturing methods such as standing culture, test tube culture, shaking culture, aeration spinner culture or fermentation. Preferably, the recombinant host cells are cultured in a fermentor. Fermentation processes of the invention include batch, fed-batch and continuous methods of fermentation. A variety of such processes have been developed and are well known in the art.

The recombinant host cells are preferably cultured under controlled pH. In one embodiment, recombinant host cells may be cultured at a pH of between 4.5 and 8.5, preferably 6.0 and 8.5, more preferably at a pH of about 7. The desired pH may be maintained by any method known to those skilled in the art.

Preferably, the recombinant host cells are further cultured under controlled aeration and under controlled temperatures. In one embodiment, the controlled temperatures include temperatures between 15 and 70° C., preferably the temperatures are between 20 and 55° C., more preferably between 30 and 50° C. The appropriate conditions are usually selected based on the choice of the expression host and the protein to be produced.

In a specific embodiment, the enzyme is expressed in Bacillus strain GX4935 (see examples). The strain is cultivated under aerobic conditions in a suitable fermentation medium. A suitable medium medium may contain assimilable sources of carbon and nitrogen besides inorganic salts optionally together with growth promoting nutrients, such as yeast extract. Fermentation is typically conducted at 35-40° C. and at a pH of 6.5-7.5 and preferably kept approximately constant by automatic means. The enzyme is excreted into the medium. At the end of fermentation, if required, the production host may be killed by means known by the person skilled in the art. The ensuing fermentation broth may be freed of bacterial cells, debris therefrom together with other solids, for example by filtration or centrifugation. The filtrate or supernatant containing the enzyme may be further clarified, for example by filtration or centrifugation, and then concentrated as required, for example by ultrafiltration or in an evaporator under reduced pressure to give a concentrate which, if desired, may be taken to dryness, for example by lyophilization or spray-drying.

After fermentation, if necessary, the cells can be removed from the fermentation broth by means of centrifugation or filtration. After fermentation has stopped or after removal of the cells, the enzyme of the invention may then be recovered and, if desired, purified and isolated by conventional means, including, but not limited to, treatment with a conventional resin, treatment with a conventional adsorbent, alteration of pH, solvent extraction, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilisation and the like. For example, the enzymes according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art (Protein Purification Protocols, Methods in Molecular Biology series by Paul Cutler, Humana Press, 2004). Usually, the compound is “isolated” when the resulting preparation is substantially free of other components.

In an embodiment, an isolated enzyme preparation is provided having a purity of about 80% (by dry weight) of the enzyme of the invention or more (i.e. less than about 20% of all the media, components or fermentation byproducts). In a specific embodiment, the invention provides the enzyme of the invention in a purity of about 90% or more, preferably in a purity of 95% or more, in particular in a purity of 98% or more. In practice, a minor amount of other components may be present in an isolated enzyme preparation of the invention. Thus, a purified preparation of the enzyme may comprise 99% or less of the enzyme, in particular 98% or less.

Alternatively, however, the enzyme of the invention is not purified from the recombinant host cell or the culture. The entire culture or the culture supernatant may be used as a source of the enzyme. In a specific embodiment, the culture or the culture supernatant comprising the enzyme is used without substantial modification.

-   -   It is further noted that it is also possible to make the enzyme         of the invention, such as the subtilisin BPN′ variant, by known         chemical protein synthesis technology, e.g. by solid phase         peptide synthesis. However, expression of the subtilisin mutants         in microbial host cells will generally be preferred since this         will allow for the microbial host cell to produce the subtilisin         protein in a proper conformation for enzymatic activity.         However, it should be possible to convert improperly folded         subtilisin BPN′ variants or homologues thereof into an active         conformation.

The enzymes of the invention (subtilisin BPN′ variants or homologues thereof) may be chemically or biochemically modified, e.g. post-translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of a tag, as already mentioned above. Such modified polypeptides and proteins fall within the scope of the term “enzyme” of the invention.

In order to further illustrate the present invention and the advantages thereof, the following specific examples are given, it being understood that the same is intended only as illustrative and in nowise limitative.

EXAMPLES Production of Enzymes (for Use) According to the Invention Mutagenesis, Cloning and Expression

The gene coding for subtilisin B S149 (Ruan et al. 2008) was obtained from Philip N. Bryan (University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Md. 20850). Mutagenesis was performed using a pUB110 based Escherichia. coli-Bacillus subtilis (E. coli-B. subtilis) shuttle vector harboring the BS149 gene using either the native promotor or alternatively using the aprE promotor and optionally a C-terminal his-tag (pBE-S DNA, http://www.clontech.com/takara). The gene encoding an enzyme according to the invention was constructed by introducing the mutations S221C and P225A into the BS149 gene using the site-directed mutagenesis method (Sambrook et al.,1989). All primers were designed using the Agilent Primer design tool (http://www.genomics.agilent.com). The constructed sequences were verified by DNA sequencing before transformation to Bacillus subtilis GX4935.

In order to produce BS149-DM without a His-tag the gene coding for BS149-DM and its natural promoter sequence was cloned into the pBS42 shuttle vector (DSMZ, Germany) at EcoRI/BamHI sites. The ligation mixtures were transformed into competent Escherichia coli and transformants were plated on LB plates containing chloramphenicol (34 μg/mL). The plasmid pBS42-S5 was propagated in E. coli, isolated and validated by sequencing. The sequence validated plasmid was used to transform B. subtilis host.

The gene coding for BS149-DM with a His-tag was cloned into a pUB-110 based E. coli-B. subtilis shuttle vector (pBES) using the MluI and BamHI site (FIG. 12). The polynucleotide sequence of a gene (BS149-DM) encoding an enzyme (polypeptide) of the invention and the encoded enzyme is shown in SEQUENCE ID NO 5. The corresponding amino acid sequence is numbered according to the subtilisin BPN′ numbering scheme. Amino acids-107 to -1 comprise the signal sequence, the pre sequence and a pro sequence which are cleaved off upon full maturation. Amino acids 1-275 comprise the mature enzyme which exhibits the full catalytic activity. In order to enable a fast and efficient purification after amino acid 275 a C-terminal His-tag is attached as shown in SEQUENCE ID NO 5. As a consequence of the removal of a calcium binding site BS149-DM contains a deletion of 9 amino acids compared to subtilisin BPN′ comprising the amino acids corresponding to L75, N76, N77, S78, I79, G80, V81, L82 and G83 in subtilisin BPN′. In order to maintain the subtilisin BPN′ numbering for BS149-DM the numbering jumps from 74 to 83. In the shuttle vector, the expression of the gene is under the control of aprE promoter. The vector contained the pUB ori of replication for Bacillus and a kanamycin resistance marker. The vector also contained the ColE1 ori of replication and an ampicillin resistance marker for maintenance in E. coli. The resulting plasmid pBES-BS149DMHIS was propagated in E. coli TOP10 and transformed into B. subtilis GX4935 (ΔnprEΔaprE).). Using pBES-BS149DMHIS as the template, mutagenesis was carried out by the Quikchange method (Agilent). Alternatively other methods for site directed mutagenesis known in the art may be used (Sambrook et al., 1989.).

The gene of Subtiligase (Abrahmsén et al. 1991) was ordered at DNA2.0 (https://www.dna20.com/) in a DNA2.0 pJ201 cloning vector and recloned into E. coli-B. subtilis shuttle vector (pBS42 DSM 8748 obtained from DSMZ; pBS42-S5). The pJ201 vector (DNA 2.0) harboring Subtiligase as well as the pBS42 shuttle vector (DSMZ) were digested with EcoRI and BamHI (NEB). Linearized shuttle vector as well as Subtiligase insert were isolated from gel and ligated (LigaFast, Promega). The construct was transformed to E. coli MM294 strain (DSMZ). The plasmid pBS42-S5 was propagated in E. coli, isolated and validated by sequencing (FIG. 13). The validated DNA was used for transformation of either B. subtilis DB104 or B. subtilis GX4935. The B. subtilis GX4935 strain has reduced extracellular proteolytic activity (Kawamura and Doi 1984; Fahnestock and Fisher 1987). The addition of the wild type subtilisin to promote production of the mature form as reported by Abrahmsén et al. 1991 was not necessary.

Except for subtiligase and for the BS149-DM without His-tag in Example 23, in all experiments, enzymes were prepared making use of a C-terminal His-tag.

Production and Purification of Synthetic Subtilisin BPN′ Variants

Transformants in pBS42 shuttle vector were picked and grown on LB plate containing 10 μg/mL chloramphenicol at 37° C. for 16 h, were picked and inoculated into 5 mL of LB broth containing 10 μg/mL chloramphenicol. After 16 h of incubation at 37° C., 1% (v/v) of the cultures were inoculated to 1 liter terrific broth (12 g/l tryptone, 24 g/l yeast extract, 0.4% (v/v) glycerol, 17 mM KH₂PO₄ and 72 mM K₂HPO₄, 50 mg/L Trp, 50 mg/L Lys, 50 mg/L Met). Cultures were grown at 37° C. with vigorous shaking and incubation was continued for 48 hours. After 48 h expression, cells were isolated from the medium by centrifugation at 6,000 g for 20 min, 4° C. Subsequently, 5 g of CaCl₂ were added to the medium and the pH was adjusted back to 7.5. The precipitate was pelleted by centrifugation at 6,000 g for 20 min, 4° C.

Ammoniumsulfate was added to the supernatant to a final concentration of 45% (w/v) to precipitate the enzyme. The precipitated enzyme was harvested by centrifugation at 8,000 g for 40 min, 4° C. The pellet was washed with 80% acetone, and resuspended in 15 mL water. The protein sample was desalted using a HiPrep 26/10 desalting column (GE healthcare) in buffer (20 mM Tricine, 1 mM CaCl₂ pH 7.5). The desalted proteins were loaded on a HiTrap Q HP column (GE healthcare). Flow through, which contains the enzyme, was collected and concentrated. The purity of the protein was analyzed by SDS-PAGE and enzyme concentration was determined by measuring the absorbtion at 280 nm (Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990) e.g. by NanoDrop spectrophotometer (Thermo Fisher Scientific Inc). The specific extinction coefficient can be calculated at http://web.expasy.org/protparam/according to Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., Appel R. D., Bairoch A.;

Protein Identification and Analysis Tools on the ExPASy Server;

(In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). pp. 571-607. Purity of about 90% or more was feasible. The obtained enzyme was provided at a concentration of about 2 mg/mL in an aqueous solution in 20 mM Tricine, 1 mM CaCl₂ pH 7.5. This enzyme solution was used as such for the (oligo)peptide fragment condensations and cyclisations.

Production and Purification of Synthetic Subtilisin BPN′ Variants which Carry a His-Tag

A single microbial colony of B. subtilis containing a plasmid with the subtilisin variant gene of interest was inoculated in 5 mL LB with kanamycin (10 μg/mL) at 37° C. in a shaking incubator. To the 30 mL Terrific Broth supplemented with antibiotic (kanamycin 10 μg/mL) and amino acids (100 mg/L Trp, 100 mg/L Met and 100 mg/L Lys) 0.6 mL of the overnight culture was added. The cells were grown 48 h at 37° C. in a shaking incubator (200 rpm). The cells were harvested by centrifugation (15 min, 4,000 rpm, 4° C.). The medium (30 mL) was decanted and concentrated on Amicon-centrifugal unit (15 ml, 10 kDa MW cut-off) in two centrifugation steps (15 min, 4000 rpm, 4° C.). The concentrated medium (0.5 ml) was then exchanged for buffer A (25 mM Tricine, pH 7.5, 0.5M NaCl, 20 mM imidazole) in three washing/concentrating steps (14 ml buffer A, 10 min, 4,000 rpm, 4° C.). For His-tag purification Talon resin (2.5 ml, Clonetech) was added to a plastic column cartridge. The resin was washed with 5 mL MilliQ water and equilibrated with 5 mL of buffer A. The crude enzyme was loaded on the column and washed with 5 mL buffer A. The enzyme was eluted with 5 mL buffer B (25 mM Tricine, pH 7.5, 0.5M NaCl, 200 mM imidazole). The elute was concentrated on a Amicon-centrifugal unit (5 ml, 10kDa MW cut-off) by centrifugation (15 min, 4000 rpm, 4° C.) and the buffer was exchanged to 25 mM Tricine, pH 7.5 in three washing/concentrating steps (5 ml buffer, 10 min, 4, 000 rpm, 4° C.).

The purity and enzyme concentration was determined as described above Purity was more than 90%, The obtained aqueous solution (25 mM Tricine, pH 7.5) containing about 2 mg/ml of the obtained enzyme was used as such for the (oligo)peptide fragment condensations and cyclisations.

References

Abrahmsén, L, J Tom, J Burnier, K A Butcher, A Kossiakoff, and J A Wells. 1991. “Engineering Subtilisin and Its Substrates for Efficient Ligation of Peptide Bonds in Aqueous Solution.” Biochemistry 30 (17) (April 30): 4151-9. http://www.ncbi.nlm.nih.gov/pubmed/2021606.

Fahnestock S R, Fisher K E: Expression of the staphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene. J Bacteriol. 1986 March; 165(3):796-804

Kawamura, Fujio, and Roy H. Doi. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 October; 160(1):442-4

Ruan, Biao, Viktoriya London, Kathryn E Fisher, D Travis Gallagher, and Philip N Bryan. Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant. Biochemistry. 2008 Jun. 24; 47(25):6628-36.

Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Wells, James A, Eugenio Ferrari, Dennis J Henner, David A Estell, and Ellson Y Chen.

Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis.

Nucleic Acids Res. 1983 Nov. 25; 11(22):7911-25.

Enzymatic Fragment Condensation and Cyclisation Examples Materials and Methods

Unless stated otherwise, chemicals were obtained from commercial sources and used without further purification. Analytical HPLC was performed on an HP1090 Liquid Chromatograph, using a reversed-phase column (Phenomenex, C18, 5 μm particle size, 150×4.6 mm) at 40° C. UV detection was performed at 220 nm using a UV-VIS 204 Linear spectrometer. The gradient program was: 0-25 min linear gradient ramp from 5% to 98% eluent B and from 25.1-30 min 5% eluent B (eluent A: 0.5 mL/L methane sulfonic acid (MSA) in H2O, eluent B 0.5 mL/L MSA in acetonitrile). The flow was 1 mL/min from 0-25.1 min and 2 mL/min from 25.2-29.8 min, then back to 1 mL/min until stop at 30 min. Injection volumes were 20 μL. Preparative HPLC was performed on a Varian PrepStar system using a stationary-phase column (Pursuit XRs, C18, 10 μm particle size, 500×41.4 mm). LC-MS was performed on an Agilent 1200 series Liquid Chromatograph, using a reversed-phase column (Phenomenex, C18, 5 μm particle size, 150×4.6 mm) at 40° C. UV detection and gradient program were as described for analytical HPLC. The molecular weights were determined using an Agilent 6130 quadrupole LC/MS system.

Protocol 1: N-Fmoc-Protected (Oligo)Peptide-OCam Esters were Synthesized as Described Below

1 gram of Rink resin (4-((2,4-dimethoxyphenyl)(Fmoc amino)methyl)phenoxyalkyl linker, with a loading of 0.64 mmol/gram) was washed with dichloromethane (DCM, 2×2 min, 10 mL) and 1-methyl-2-pyrrolidone (NMP, 2×2 min, 10 mL) and Fmoc-deprotected using piperidine/NMP (1/4, v/v, 2×8 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), iodoacetic acid (4 equiv.) was coupled to the resin using DCC (4 equiv.) and HOAt (4 equiv.) in DCM (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and THF (2×2 min, 10 mL), the resin was loaded with an Fmoc-protected amino acid using 4 equiv. Fmoc-Xxx-OH and 10 equiv. DiPEA in DMF/THF (1/1, v/v, 10 mL) at 50° C. for 20 h. Here and in other parts of this disclosure ‘Xxx’ stands for one amino acid (variable as indicated in the Figures belonging to the examples below).

After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), standard SPPS protocols were followed to elongate the peptide (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage from the resin and side-chain deprotection was performed using a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide was precipitated using methyl tent-butyl ether (MTBE)/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 2: N-Fmoc-Protected (Oligo)Peptide-OCam-Xxx-NH₂ Esters were Synthesized as Described Below

1 gram of Rink resin was washed with DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and Fmoc-deprotected using piperidine/NMP (1/4, v/v, 2×8 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), Fmoc-Xxx-OH (4 equiv.) was coupled to the resin using HBTU (4 equiv.), HOBt (4 equiv.) and DiPEA (8 equiv.) in NMP (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), the resin was Fmoc-deprotected using piperidine/NMP (1/4, v/v, 2×8 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), iodoacetic acid (4 equiv.) was coupled using DCC (4 equiv.) and HOAt (4 equiv.) in DCM (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and THF (2×2 min, 10 mL), an Fmoc-protected amino acid was coupled using 4 equiv. Fmoc-Xxx-OH and 10 equiv. DiPEA in DMF/THF (1/1, v/v, 10 mL) at 50° C. for 20 h. After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), standard SPPS protocols were followed to elongate the peptide (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 3: N-Fmoc-Protected (Oligo)Peptide-OCam-Xxx-OH Esters were Synthesized as Described Below

1 gram of Trityl resin (2-chloro-chlorotrityl linker, with a loading of 1.0 mmol/gram) was washed with DCM (2×2 min, 10 mL) and Fmoc-Xxx-OH (2 equiv.) was coupled to the resin using DiPEA (5 equiv.) in DCM (30 min, 10 mL). After washing with DMF (2×2 min, 10 mL), the unreacted chlorotrityl groups were capped using DCM/MeOH/DiPEA (80/15/5, v/v/v, 2×10 min, 10 mL). The resin was washed with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and Fmoc-deprotected using piperidine/NMP (1/4, v/v, 2×8 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), iodoacetic acid (4 equiv.) was coupled using DCC (4 equiv.) and HOAt (4 equiv.) in DCM (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and THF (2×2 min, 10 mL), an Fmoc-protected amino acid was coupled using 4 equiv. Fmoc-Xxx-OH and 10 equiv. DiPEA in DMF/THF (1/1, v/v, 10 mL) at 50° C. for 20 h. After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL), standard SPPS protocols were followed to elongate the peptide (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 4: (Oligo)Peptide C-Terminal Amide Nucleophiles were Synthesized as Described Below

1 gram of Rink resin (4-((2,4-dimethoxyphenyl)(Fmoc-amino)methyl)-phenoxyalkyl linker, with aloading of 0.64 mmol/gram) was washed with DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and Fmoc-deprotected using piperidine/NMP (1/4, v/v, 2×8 min, 10 mL). Standard SPPS protocols were followed to elongate the peptide (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 5: N-Acetyl-Protected (Oligo)Peptide Activated Esters were Synthesized as Described Below

After SPPS of the desired sequence according to one of the protocols 1-3, the resin bound peptide was Fmoc-deprotected using piperidine/NMP (1/4, v/v, 2×8 min, 10 mL). The resin was washed with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and the peptide N-terminal amine function was acetylated using a mixture of Ac₂O (10 vol %), DiPEA (5 vol %), HOBt (0.2 wt %) in NMP (2×10 min, 10 mL). The resin was washed with NMP (3×2 min, 10 mL) and DCM (3×2 min, 10 mL). Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 6: (Oligo)Peptide C-Terminal Acids were Synthesized as Described Below

1 gram of Trityl resin (2-chloro-chlorotrityl linker, with a loading of 1.0 mmol/gram) was washed with DCM (2×2 min, 10 mL) and Fmoc-Xxx-OH (2 equiv.) was coupled to the resin using DiPEA (5 equiv.) in DCM (30 min, 10 mL). After washing with DMF (2×2 min, 10 mL), the unreacted chlorotrityl groups were capped using DCM/MeOH/DiPEA (80/15/5, v/v/v, 2×10 min, 10 mL). The resin was washed with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and NMP (2×2 min, 10 mL) and standard SPPS protocols were followed to elongate the peptide (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Protocol 7: Synthesis Partially Protected (Oligo)Peptide Fragments

During SPPS of the peptide sequence according to one of the protocols 1-6, at the desired position a differently (TFA stable) protected amino acid was coupled such as Fmoc-Asp(OcHex)-OH, Fmoc-Glu(OBn)-OH or Fmoc-Lys(Alloc)-OH. Cleavage from the resin and side-chain deprotection, except for the TFA stable cHex, Bn or Alloc group which remained unaffected, was performed using a mixture of TFA/TIS/water (95/2.5/2.5, v/v/v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE/n-heptanes (1/1, v/v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE/n-heptanes (1/1, v/v, 50 mL) followed by lyophilization from acetonitrile/water (1/1, v/v, 50 mL).

Coupling Examples

Note: The enzyme denoted as BS149-DM (SEQUENCE ID NO:5) contains a deletion of amino acids 75-83 and mutations Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, S221C, P225A, T254A and Q271E compared to SEQUENCE ID NO:2. On the basis of the present disclosure, common general knowledge and optionally a limited amount of route testing, the skilled person in the art may revert one or more of mutations Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, T254A and Q271E or make different substitutions at one or more of the positions Q2, S3, P5, S9, 131, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218S, T254, Q271 while still having significantly improved properties compared to Subtiligase (see for instance example 24).

The enzyme denoted as Subtiligase contains the mutations S221C and P225A compared to SEQUENCE ID NO:2.

The enzymes of the invention used in the Examples 1-23 have all the mutations of BS149-DM, plus optional additional mutations as mentioned in the Examples.

As indicated below, enzymes with further mutations were made using the technology described above.

Example 1: Enzymatic Oligopeptide Fragment Coupling using Different BS149-DM Mutants

To test the activity and S/H ratio of the different mutants, the following standard reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5 μg enzyme was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated.

The activity of the different BS149-DM mutants is defined as the total of the amount of product and the amount of hydrolysed pentapeptide C-terminal Cam-ester divided by the total of the amount of product, hydrolysed pentapeptide C-terminal Cam-ester and remaining Cam-ester, within the specified reaction time. The most active mutant was set to 100% (see FIG. 1 A). The S/H ratio of the different BS149-DM mutants is defined as the amount of product divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified time (see FIG. 1B).

Activity and S/H ratio in other examples were determined in the same way, unless specified otherwise.

Conclusions: clearly, BS149-DM has a twice higher activity and an improved S/H ratio (1.8 versus 0.9) as compared to subtiligase. The M222 position proved very important for the S/H ratio of the enzyme. Especially good results were obtained with the M222G and M222P mutants of BS149-DM. All BS149-DM variants containing a P4 pocket mutation (positions Y104, I107 and L135) have a comparable S/H ratio to BS149-DM. However, for certain mutations, the activity of the BS149-DM variants was drastically improved. Particularly good results were obtained with the mutations Y104S, I107V, L135D, L135N and L135S. When combining P4 pocket mutations, BS149-DM variants with even higher activity were obtained, i.e. I107V+L135S and I107V+L135N. When P4 pocket mutations were combined with P1′ pocket mutations, a very active BS149-DM variant with an increased S/H ratio as compared to BS149-DM was obtained, e.g. I107V+M222G.

Example 2: Enzymatic Oligopeptide Fragment Coupling using Different BS149-DM+M222P+L217 Mutants

To test the activity and S/H ratio of the different mutants, the same reaction as described in Example 1 was performed. The activity of the different BS149-DM+M222P+L217 mutants is defined as the total of the amount of product and the amount of hydrolysed oligopeptide C-terminal Cam-ester divided by the total of the amount of product, hydrolysed oligopeptide C-terminal Cam-ester and remaining Cam-ester. The most active mutant was set to 100% (see FIG. 2 A). The S/H ratio of the different BS149-DM+M222P+L217 mutants is defined as the amount of product divided by the amount of hydrolysed C-terminal Cam-ester (see FIG. 2 B).

Conclusions: clearly, all BS149-DM+M222P+L217 mutants have an improved S/H and similar or improved activity as compared to subtiligase and some of them have an increased activity compared to BS149-DM+M222P. Particularly good results were obtained with the mutations L217N, L217T, L217E, L217I, L217V and L217A. The L217 position proved not only very important for activity and S/H ratio but is even more important for the substrates scope, as described in Example 5.

Example 3: Mapping the P4 Pocket Substrate Specificity of Different BS149-DM Mutants Containing a P4 Pocket Mutation (Positions Y104, I107 and L135)

To determine the P4 pocket substrate specificity of the different mutants, the following standard reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 200 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Xxx-Ser-Lys-Leu-OCam.TFA in 1.2 mL water+1 mL Acn). Couplings with all these peptide esters were performed, differing in the amino acid at this position, as indicated in FIGS. 3A-3C.

To this mixture 5.5 μg enzyme was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated. The activity is defined as the amount of product divided by the total of the amount of product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester, within the specified reaction time. The most active substrate was set to 100%, see FIGS. 3 A-C.

Conclusions: as evident from FIGS. 3A-C, the P4 substrate scope of the BS149-DM mutants with a P4 mutation (on positions Y104, I107 and/or L135) clearly differs from that of BS149-DM which may be advantageous for various particular peptide sequences. Several mutants show a much broader P4 substrate scope than BS149-DM. This is in particular the case with mutations I107V, L135D, L135N and L135S.

Example 4: Mapping the P1′ and P2′ Pocket Substrate Specificity of Different BS149-DM+M222 Mutants

To determine the P1′ and P2′ pocket substrate specificity of the different mutants, the following two standard reactions were performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Xxx-Leu-Arg-NH₂.2TFA for P1′ and H-Ala-Xxx-Arg-NH₂.2TFA for P2′ in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5 μg enzyme was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated. The activity is defined as the amount of product divided by the total of the amount of product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester, within the specified reaction time. The most active substrate was set to 100%, The P1′ and P2′ pocket substrate specificities of different BS149-DM+M222 mutants are shown in FIGS. 4 A-D. The coupling with tryptophan in P1′ position was not determined due to overlap in the LC-MS peaks.

Conclusions: as evident from FIGS. 4 A-D, the P1′ and P2′ substrate scopes of the BS149-DM mutants with a P1′ mutation on position M222 clearly differs from that of BS149-DM which may be advantageous for various particular peptide sequences. Several mutants show a much broader P1′ and P2′ substrate scope than BS149-DM. This is in particular the case with mutations M222G and M222P.

Example 5: Mapping the P1′ and P2′ Pocket Substrate Specificity of Different BS149-DM+M222P+L217 Mutants

To determine the P1′ and P2′ pocket substrate specificity of the different mutants, the same reactions and analyses were performed as described in Example 4. The P1′ and P2′ pocket substrate specificities of different BS149-DM+M222P+L217 mutants are shown in FIGS. 5 A-L.

Conclusions: as evident from FIGS. 5 A-L, the P1′ and P2′ substrate scopes of the BS149-DM+M222P mutants with a P1′ mutation on position L217 clearly differs from that of BS149-DM+M222P which may be advantageous for various particular peptide sequences. Several mutants show a much broader P1′ and P2′ substrate scope than BS149-DM. This is in particular the case with mutations L217G and L217H. Several mutants show a drastically improved activity for certain particular substrates. For instance improved activity for Phe in the P1′ pocket for mutations BS149-DM+M222P+L217N, E, G, Y, F or H. The mutant BS149-DM+M222P+L217H also shows a much increased activity for Asn in the P1′ pocket. The mutants BS149-DM+M222P+L217E and A have an improved activity for Leu, Ile and Val in the P1′ pocket. The mutants BS149-DM+M222P+L217T and S have an improved activity for Asp in the P1′ pocket.

Example 6: Mapping the P1′ Pocket Substrate Specificity of Different BS149-DM+M222G+L217 Mutants

To determine the P1′ pocket substrate specificity of the different mutants, the same reactions and analyses were performed as described in Example 4. The P1′ pocket substrate specificities of different BS149-DM+M222G+L217 mutants are shown in FIGS. 6A-F.

Conclusions: as evident from FIGS. 6A-F, the P1′ substrate scopes of the BS149-DM+M222G mutants with a P1′ mutation on position L217 clearly differs from that of BS149-DM+M222G which may be advantageous for various particular peptide sequences. Several mutants show a much broader P1′ substrate scope than BS149-DM. This is in particular the case with mutations L217G and L217F. Several mutants show a drastically improved activity for certain particular substrates. For instance improved activity for Phe in the P1′ pocket for mutations BS149-DM+M222G+L217N, E, G, Y, F, I or H. The mutant BS149-DM+M222G+L217F also shows a much increased activity for Asn in the P1′ pocket. The mutants BS149-DM+M222G+L217F, G, A and Y have an improved activity for Leu, Ile and Val in the P1′ pocket. The mutants BS149-DM+M222G+L217R, T and S have an improved activity for Asp in the P1′ pocket.

Example 7: Mapping the P1′, P2′ and P4 Pocket Substrate Specificity of BS149-DM+M222G+I107V Mutant

To determine the P1′ and P2′ pocket substrate specificity of BS149-DM+M222G+I107V, the same reactions and analyses were performed as described in Example 4. The P1′ and P2′ pocket substrate specificities of the BS149-DM+M222G+I107V mutant are shown in FIGS. 67A and B, respectively. To determine the P4 pocket substrate specificity of BS149-DM+M222G+I107V, the same reactions and analyses were performed as described in Example 3. The P4 pocket substrate specificity of the BS149-DM+M222G+I107V mutant is shown in FIG. 7C.

Conclusions: as evident from FIGS. 7A-C, the P1′ and P2′ substrate scope as well as the P4 substrate scope of the BS149-DM+M222G+I107V mutant are broader as compared to BS149-DM. Clearly, the advantageous mutations for the P1′ and P2′ pockets (i.e. M222G) and for the P4 pocket (i.e. I107V) can be successfully combined since the substrate broadness is comparable to the BS149-DM+M222G mutant but the S/H ratio is significantly higher (see Example 1).

Example 8: Enzymatic Coupling Reactions using Different N-Acetyl Protected Oligopeptide C-Terminal Cam-Ester Acyl Donors

Peptide ligation reactions were performed at 25° C. in 100 mM Tricine buffer (pH 8.0), containing 15 μM BS149-DM, 10 mM peptide C-terminal Cam-ester (Ac-Asp-Leu-Ser-Lys-Gln-OCam.TFA, Ac-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA, Ac-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA or Ac-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA) and 15 mM dipeptide C-terminal amide (H-Ala-Phe-NH₂). After 180 min the reaction mixtures were analyzed by LC-MS. The product, hydrolysed C-terminal Cam-ester and remaining Cam-ester peaks were integrated. The S/H ratio of the different reactions is defined as the amount of product divided by the amount of hydrolysed C-terminal Cam-ester, within the specified reaction time.

TABLE 1 Coupling of different acyl donors with H-Ala-Phe-NH₂ Peptide amine S/H Peptide C-terminal Cam-ester nucleophile ratio Ac-Asp-Leu-Ser-Lys-Gln-Ocam H-Ala-Phe-NH₂ 12 Ac-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Ocam H-Ala-Phe-NH₂ 59 Ac-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam H-Ala-Phe-NH₂ 41 Ac-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam H-Ala-Phe-NH₂ 80

Conclusions: different lengths of oligopeptide acyl donors can be used. The S/H ratio increases with the length of the oligopeptide acyl donor.

Example 9: Enzymatic Coupling Reactions using Different Oligopeptide C-Terminal Amide Nucleophiles

Peptide ligation reactions were performed at 25° C. in 100 mM Tricine buffer (pH 8.0), containing 15 μM BS149-DM, 1 mM pentapeptide C-terminal Cam-ester (Ac-Phe-Ile-Glu-Trp-Leu-OCam) and 3 mM peptide amine nucleophile (H-Ala-Phe-NH₂, H-Ala-Phe-Ala-NH₂ or H-Ala-Phe-Ala-Tyr-NH₂). After 60 min the reaction mixtures were analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated. The S/H ratio of the different reactions is defined as the amount of product divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified reaction time.

TABLE 2 Coupling of different oligopeptide nucleophiles with Ac-Phe-Ile-Glu-Trp-Leu-OCam Peptide C-terminal Cam-ester Peptide amine nucleophile S/H ratio Ac-Phe-Ile-Glu-Trp-Leu-OCam H-Ala-Phe-NH₂ 1.5 Ac-Phe-Ile-Glu-Trp-Leu-OCam H-Ala-Phe-Ala-NH₂ 1.7 Ac-Phe-Ile-Glu-Trp-Leu-OCam H-Ala-Phe-Ala-TyrNH₂ 1.9

Conclusions: different lengths of oligopeptide nucleophiles can be used. The S/H ratio increases with the length of the oligopeptide nucleophile.

Example 10: Effect of the pH on the S/H Ratio of BS149-DM+M222G Mutant

To examine the effect of pH on the S/H ratio of the BS149-DM+M222G mutant, the following standard reaction was performed. 800 μL of phosphate buffer (1M, pH 7.0-8.8), or tricine buffer (1M, pH 7.9-8.9) or carbonate buffer (1M, pH 9.2-10.6) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water). To this mixture 5.5 μg enzyme was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated and the S/H ratio is defined as the amount of product divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified reaction time, see FIG. 8.

Conclusions: the S/H ratio of BS149-DM+M222G is dependent on the pH and there is a clear optimum between pH 8 and pH 9, but lower or higher pH can also be used depending on the solubility and stability properties of the oligopeptides.

Example 11: Effect of the Concentration of Acyl Donors and Nucleophiles on the S/H Ratio of BS149-DM+M222G

To examine the effect of substrate concentration on the S/H ratio of mutant BS149-DM+M222G, the following reactions were performed. A stock solution of tripeptide C-terminal amide (12.9 mg H-Glu-Leu-Arg-NH₂.2TFA or 11.7 mg H-Ala-Leu-Arg-NH₂.2TFA) and C-terminal pentapeptide Cam-ester (4.2 mg Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA) in 150 μL water was prepared. The mixtures were brought to neutral pH with 5.1 μL NaOH (32wt % in water). To prepare reaction mixtures with different concentration of substrates, 10 μL of one of the above stock solutions was diluted with 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10000 μL phosphate buffer (1M, pH 8.5). To these reaction mixtures 11 μg of BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated and the S/H ratio is defined as the amount of product divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified reaction time, see FIGS. 9A and B.

Conclusions: the S/H ratio is dependent on the substrate concentrations. There is an optimal substrate concentration for each individual substrate depending on the affinity of the nucleophile for the enzyme and on the solubility and stability properties of the oligopeptides.

Example 12: Effect of Dosing of the Acyl Donor on the S/H Ratio using BS149-DM+M222G

To examine the effect of dosing of the acyl donor on the S/H ratio of mutant BS149-DM+M222G, the following two reactions were performed. In two-fold, 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 5.5 μg BS149-DM+M222G. To one of these mixtures, 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water) was added and the reaction mixture was shaken (150 rpm) at room temperature. To the other mixture 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water) was dosed in portions of 3.5 μL every minute while shaking the reaction mixture (150 rpm) at room temperature. After 30 min a 550 μL aliquot of both reaction mixtures was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. For both reactions the conversion of the pentapeptide C-terminal Cam-ester starting material was 100%. The product and hydrolysed pentapeptide C-terminal Cam-ester peaks were integrated and the S/H ratio is defined as the amount of product divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified reaction time. The S/H ratio for the reaction where all acyl donor was added at t=0 was 2.45 and the S/H ratio for the reaction where the acyl donor was dosed every minute was 2.73.

Conclusions: by dosing the oligopeptide C-terminal Cam-ester in time the S/H ratio can be improved.

Example 13: Cyclization of Oligopeptide C-Terminal Cam-Esters using Different Enzymes

The following experiments were performed to determine the S/H ratio of Subtiligase, BS149-DM and BS149-DM+M222G for the cyclization of an oligopeptide C-terminal Cam-ester,.

800 μL of phosphate buffer (100 mM, pH 8.0) was added to a 100 μL stock solution of an oligopeptide C-terminal Cam-ester with an N-terminal free amine (0.01 mmol H-Ala-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-Tyr-Gln-Ser-Tyr-OCam.2TFA in 1 mL water) containing 5 mg/mL dithiotreitol. To this mixture 5.5 μg enzyme was added and the reaction mixtures were shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixtures was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed C-terminal Cam-esterand remaining Cam-ester starting material peaks were integrated and the S/H ratio of the different enzymes is defined as the amount of product divided by the amount of hydrolysed C-terminal Cam-ester, within the specified reaction time, see FIG. 10.

Conclusions: evidently, also for peptide cyclisation, BS149-DM has an improved S/H ratio as compared to Subtiligase. The BS-149-DM+M222G mutant has an even higher S/H ratio.

Example 14: Effect of the pH on the S/H Ratio During Cyclisation of an Oligopeptide C-Terminal Cam-Ester using BS149-DM+M222G

To determine the effect of the pH on the S/H ratio of BS149-DM+M222G during the cyclisation of an oligopeptide C-terminal Cam-ester, the following standard reactions were performed. 800 μL of phosphate buffer (1M, pH 5, 6, 7, 8 and 9) was added to 100 μL stock solution of a tridecapeptide C-terminal Cam-ester with an N-terminal free amine (0.01 mmol H-Ala-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-Tyr-Gln-Ser-Tyr-OCam.2TFA in 1 mL water) containing 5 mg/mL dithiotreitol. To this mixture 5.5 μg BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed C-terminal Cam-ester and remaining C-terminal Cam-ester starting material peaks were integrated and the S/H ratio is defined as the amount of product divided by the amount of hydrolysed C-terminal Cam-ester, within the specified reaction time, see FIG. 11.

Conclusions: the S/H ratio of BS149-DM+M222G used for enzymatic oligopeptide cyclisation is dependent on the pH, albeit to a lesser extent than for enzymatic oligopeptide fragment condensation.

Example 15: Fragment Condensation with Oligopeptides Over 10 Amino Acids Long

To examine whether enzymatic fragment condensation with longer oligopeptides in aqueous solution is feasible, the following standard reaction was performed. 800 μL of phosphate buffer (1M, pH 8.0) was added to a mixture of 100 μL decapeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Met-Lys-Tyr-Asn-Ser-Thr-Glu-Val-NH₂.2TFA in 300 μL water) and 200 μL tridecapeptide C-terminal Cam-ester stock solution (0.01 mmol Fmoc-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.2TFA in 1.2 mL water+1 mL DMF). To this mixture 5.5 μg BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed C-terminal Cam-ester and remaining C-terminal Cam-ester peaks were integrated. The amount of product (Fmoc-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Ala-Leu-Met-Lys-Tyr-Asn-Ser-Thr-Glu-Val-NH₂), within the specified reaction time, was 68 area %.

Conclusion: Fragment condensations with longer peptides are well-feasible.

Example 16: Fragment Condensation using Oligopeptides without N- or C-Terminal Protecting Group

To examine whether enzymatic fragment condensation without N- or C-terminal protecting group is feasible without significant side-product formation, the following standard reaction was performed. 800 μL of phosphate buffer (1M, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal carboxylic acid stock solution (0.01 mmol H-Ala-Leu-Arg-OH.2TFA in 300 μL water) and 100 μL N-terminal free amine pentapeptide C-terminal Cam-ester stock solution (0.01 mmol H-His-Ala-Glu-Gly-Thr-OCam.TFA in 1.2 mL water). To this mixture 5.5 μg of BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated. The amount of product (H-His-Ala-Glu-Gly-Thr-Ala-Leu-Arg-OH), within the specified reaction time, was 74 area %. No side-products were observed indicating that no side-reactions had occurred at the C-terminal carboxylic acid function of H-Ala-Leu-Arg-OH.2TFA nor at the N-terminal amine function of H-His-Ala-Glu-Gly-Thr-OCam.

Conclusion: some oligopeptide sequences can be successfully enzymatically ligated without using N- or C-terminal protecting groups.

Example 17: Fragment Condensations using Oligopeptide C-Terminal Cam-Xxx-NH₂ or Cam-Xxx-OH Esters:

To examine whether enzymatic fragment condensations with Cam-Xxx-NH₂ or Cam-Xxx-OH esters are feasible, the following standard reactions were performed. 800 of phosphate buffer (1M, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA, Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-NH₂.TFA, Ac-Asp-Phe-Ser-Lys-Leu-OCam-Lys-NH₂.2TFA or Ac-Asp-Phe-Ser-Lys-Leu-OCam-Glu-NH₂.TFA in 1.2 mL water). To each of these 4 mixtures 5.5 μg of BS149-DM+M222G was added and the reaction mixtures were shaken (150 rpm) at room temperature. After 30 min a 550 μL aliquot of the reaction mixtures was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining tetrapeptide C-terminal Cam-ester peaks were integrated. The amount of product (Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH₂), within the specified reaction time, was 86 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH, 83 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-NH₂, 78 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Lys-NH₂ and 83 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Glu-NH₂.

Conclusions: this example shows that Cam-Xxx-NH₂ and Cam-Xxx-OH esters can be used successfully for enzymatic oligopeptide fragment condensation.

Example 18: Fragment Condensation using a C-Terminal Oligopeptide Thioester and BS149DM+I107V+M222G

To examine whether enzymatic oligopeptide fragment condensations using C-terminal thioesters are feasible, the following standard reaction was performed. 1 mL of Tricine buffer (100 mM, pH 7.5), containing 2.5 mM pentapeptide C-terminal thio-ester (Suc-Ala-Ala-Pro-Phe-SBz1), 25 mM dipeptide C-terminal amide (H-Gly-Phe-NH₂), and 5 μg BS149-DM+L107V+M222G, was shaken (150 rpm) at 25° C. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed tetrapeptide C-terminal thio-ester and remaining tetrapeptide C-terminal thio-ester peaks were integrated. The amount of product (Suc-Ala-Ala-Pro-Phe-Gly-Phe-NH₂), within the specified reaction time, was 85 area %.

Conclusions: this example shows that oligopeptide C-terminal thioesters can be used successfully for enzymatic oligopeptide fragment condensation.

Example 19: Fragment Condensation using a C-Terminal Oligopeptide Alkyl Ester and BS149-DM+I107V+M222G

To examine whether enzymatic oligopeptide fragment condensations using C-terminal alkyl esters are feasible the following standard reaction was performed. 1 mL of Tricine buffer (100 mM, pH 7.5), containing 2.5 mM pentapeptide C-terminal alkyl ester (Ac-Asp-Phe-Ser-Lys-Leu-OTFE (TFE=2,2,2-trifluoroethyl)), 25 mM tripeptide C-terminal amide (H-Ala-Leu-Arg-NH₂), and 5 μg BS149-DM+I107V+M222G, was shaken (150 rpm) at 25° C. After 30 min a 550 μL aliquot of the reaction mixture was withdrawn and quenched with 50 μL MSA and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal alkyl-ester and remaining pentapeptide alkyl-ester peaks were integrated. The amount of product (Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH₂), within the specified reaction time, was 55 area %.

Conclusions: this example shows that oligopeptide C-terminal alkyl esters can be used successfully for enzymatic oligopeptide fragment condensation.

Example 20: Enzymatic Oligopeptide Fragment Condensation using Partial Side-Chain Protection

To demonstrate that partial P1′ side-chain protection can be beneficial, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Asp-Leu-Arg-NH₂.2TFA or 0.01 mmol H-Asp(OcHex)-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5 μg BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining peptapeptide Cam-ester peaks were integrated. The amount of product using the unprotected substrate (H-Asp-Leu-Arg-NH₂), within the specified reaction time, was 18 area %, the amount of product using the partially side-chain protected substrate (H-Asp(OcHex)-Leu-Arg-NH₂), within the specified reaction time, was 73 area %.

To demonstrate that partial P2′ side-chain protection can be beneficial, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Asp-Glu(OBn)-Arg-NH₂.2TFA or 0.01 mmol H-Asp-Glu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5 μg BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining peptapeptide Cam-ester peaks were integrated. The amount of product using the unprotected substrate (H-Asp-Glu-Arg-NH₂), within the specified reaction time, was 15 area %, the amount of product using the partially side-chain protected substrate (H-Asp-Glu(OBn)-Arg-NH₂), within the specified reaction time, was 58 area %.

To demonstrate that partial P1 side-chain protection can be beneficial, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Asp-Leu-Arg-NH₂.2TFA) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Leu-Lys-OCam.TFA or 0.01 mmol Ac-Asp-Phe-Ser-Leu-Lys(Alloc)-OCam in 1200 μL water). To this mixture 5.5 μg BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining peptapeptide Cam-ester peaks were integrated. The amount of product using the unprotected substrate (Ac-Asp-Phe-Ser-Leu-Lys-OCam.TFA), within the specified reaction time, was 5 area %, the amount of product using the partially side-chain protected substrate (Ac-Asp-Phe-Ser-Leu-Lys(Alloc)-OCam) was 84 area %.

To demonstrate that partial P4 side-chain protection can be beneficial, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA) and 100 μL tetrapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Ser-Lys-Leu-OCam.TFA or 0.01 mmol Ac-Asp(OBn)-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5 μg BS149-DM+M222G was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed tetrapeptide C-terminal Cam-ester and remaining tetrapeptide Cam-ester peaks were integrated. The amount of product using the unprotected substrate (mmol Ac-Asp-Ser-Lys-Leu-OCam.TFA), within the specified reaction time, was 32 area %, the amount of product using the partially side-chain protected substrate (Ac-Asp(OBn)-Ser-Lys-Leu-OCam.TFA) was 78 area %.

Conclusions: this example shows that partial side-chain protection can improve the yield and/or reaction rate of enzymatic oligopeptide fragment condensations

Example 21: Thermostability of BS149-DM

The fluorescence-based thermal stability assay was used to determine the apparent melting temperature of BS149-DM and subtiligase. A sample of 20 μL of protein solution in buffer (20 mM Tricine buffer, pH 7.5) and metal ions (10 mM) or EDTA (10 mM) was mixed with 5 μL of 100 times diluted Sypro Orange (Molecular Probes, Life Technologies, USA) dye in a thin wall 96-well PCR plate. The plate was sealed with Optical-Quality Sealing Tape and heated in an CFX 96 Real Time PCR System (BioRad, Hercules, Calif., USA) from 20 to 99° C. at a heating rate of 1.75° C./min. Fluorescence changes were monitored with a charge-coupled device (CCD) camera. The wavelengths for excitation and emission were 490 and 575 nm, respectively. The thermostability of the purified BS149-DM was determined as described above. The thermostability was also determined after the addition of different metal ions and chelating agents, see Table 3 below. An apparent transition temperature (T_(m)) of 66° C. was observed, indicating that the enzyme BS149-DM well preserves the thermostability from BS149. In contrast, the Tm value of Subtiligase was determined to be 59° C.

TABLE 3 Effect of metal ions (10 mM) and the chelating agent EDTA (10 mM) on the thermostability of BS149-DM. T_(m) (° C.) Control 66 Ca²⁺ 65.5 Mg²⁺ 65 Mn²⁺ 64.5 Ni²⁺ 62 EDTA 66.5

Conclusions: clearly, BS149-DM has an improved thermostability as compared to Subtiligase. The enzyme BS149-DM is also resistant to metal ions and chelating agents, since in their presence the Tm value remains virtually unaffected.

Example 22: Effect of Organic Solvents and Different Additives on BS149-DM Activity

Peptide ligation reactions were performed at 25° C. in 100 mM Tricine buffer (pH 8.0), containing 15 μM BS149-DM, 1 mM pentapeptide C-terminal Cam-ester (Ac-Phe-Ile-Glu-Trp-Leu-OCam) and 3 mM dipeptide C-terminal amide (H-Ala-Phe-NH₂). Different amounts of metal ions (10 mM), EDTA (10 mM) or organic solvent were added and after 60 min the reaction mixtures were analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining peptapeptide Cam-ester peaks were integrated. The activity of BS149-DM is defined as the total of the amount of product and the amount of hydrolysed pentapeptide C-terminal Cam-ester divided by the total of the amount of product, hydrolysed pentapeptide C-terminal Cam-ester and remaining Cam-ester, within the specified reaction time. The most reaction with the highest activity was set to 100%, see Tables 4-8.

TABLE 4 Effect of metal ions (10 mM) and the chelating agent EDTA (10 mM) on the activity of BS149-DM. Activity (%) No additive 87 Ca²⁺ 67 Mg²⁺ 87 Mn²⁺ 100 Ni²⁺ 73 EDTA 87

TABLE 5 Effect of THF on the activity of BS149-DM. Activity (%) No additive 100 10 vol % THF 60 20 vol % THF 30 30 vol % THF 10 40 vol % THF 4

TABLE 6 Effect of DMF on the activity of BS149-DM. Activity (%) No additive 100 10 vol % DMF 64 20 vol % DMF 36 30 vol % DMF 32 40 vol % DMF 18 50 vol % DMF 14

TABLE 7 Effect of DMSO on the activity of BS149-DM. Activity (%) No additive 100 10 vol % DMSO 87 20 vol % DMSO 73 30 vol % DMSO 76 40 vol % DMSO 62 50 vol % DMSO 35

TABLE 8 Effect of GndCl on the activity of BS149-DM. Activity (%) No additive 92 0.66M GndCl 100 1.32M GndCl 90 2.00M GndCl 81 2.64M GndCl 75 3.33M GndCl 67 4.00M GndCl 43

Example 23: Enzymatic Oligopeptide Fragment Coupling Using BS149-DM with and without His-Tag

To test the activity and S/H ratio of the different enzymes, the following two standard reactions were performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 water). To this mixture was added 5.5 μg BS149-DM either with or without His-tag was added and the reaction mixtures were shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixtures was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated.

The S/H ratio of B S149-DM with His-tag respectively without His-tag is defined as the amount of product (synthesized oligopeptide) divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified time. The S/H ratio for B S149 with His-tag was 1.91 and for B S149 without His-tag 1.98.

The activity of BS149-DM with and without His-tag is defined as the total of the amount of product and the amount of hydrolysed pentapeptide C-terminal Cam-ester divided by the total of the amount of product, hydrolysed pentapeptide C-terminal Cam-ester and remaining Cam-ester, within the specified reaction time. The activity of BS149-DM with His-tag was 97.3% and for BS149-DM without His-tag 98.6%.

-   -   Conclusions: the presence or absence of the His-tag has no         significant effect on the S/H ratio and the activity.

Example 24: S/H Ratio of Enzymes Corresponding to SEQ ID NO 3 with Different Mutations

To test the activity and S/H ratio of the different mutants, the following standard reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5 μg enzyme was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated.

The S/H ratio of the different mutants is defined as the amount of product divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified time (see Table 9).

TABLE 9 S/H ratio of enzymes corresponding to SEQ ID NO 3 with different mutations Amino acid at position X225 of SEQ ID NO 3 Additive mutations S/H ratio P (proline, as in wild- 1.97 type subtilisin BPN′) A 2.03 G 1.76 A N218S 2.40 A N218S, M50F 2.55 A N218S, M50F, S3C-Q206C, 2.40 Q2K, A73L, P5S A N218S, M50F, S3C-Q206C, 2.09 Q2K, A73L, P5S, Y217L

Conclusions: clearly, several enzymes corresponding to SEQ ID NO 3 (X=A) with the S221C mutation have a twofold increased S/H ratio compared to subtiligase (S/H subtiligase=0.9, see example 1). The S/H ratio remains unaffected with X=P, G or A.

Example 25: S/H Ratio of BS149-DM+M222P+L217H+X225 Mutants

To test the activity and S/H ratio of the different mutants, the following standard reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL tripeptide C-terminal amide stock solution (0.01 mmol H-Ser-Leu-Arg-NH₂.2TFA in 300 μL water) and 100 μL pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). To this mixture 5.5 μg enzyme was added and the reaction mixture was shaken (150 rpm) at room temperature. After 30 min a 500 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS. The product, hydrolysed pentapeptide C-terminal Cam-ester and remaining pentapeptide C-terminal Cam-ester peaks were integrated.

The S/H ratio of the different mutants is defined as the amount of product divided by the amount of hydrolysed pentapeptide C-terminal Cam-ester, within the specified time (see Table 10).

TABLE 10 S/H ratio of BS149-DM + M222P + L217H + X225 mutants Mutant S/H ratio BS149-DM + M222P + L217H + X225N 7.33 BS149-DM + M222P + L217H + X225D 6.69 BS149-DM + M222P + L217H + X225S 6.07 BS149-DM + M222P + L217H + X225C 5.25 BS149-DM + M222P + L217H + X225G 4.63 BS149-DM + M222P + L217H + X225A 4.47 BS149-DM + M222P + L217H + X225T 4.28 BS149-DM + M222P + L217H + X225V 4.26 BS149-DM + M222P + L217H + X225I 4.00 BS149-DM + M222P + L217H + X225L 3.55 BS149-DM + M222P + L217H + X225H 1.84 BS149-DM + M222P + L217H + X225Q 1.45 BS149-DM + M222P + L217H + X225F 0.71 BS149-DM + M222P + L217H + X225E 0.36 BS149-DM + M222P + L217H + X225P 0.17 BS149-DM + M222P + L217H + X225K 0.07 BS149-DM + M222P + L217H + X225Y 0.03 BS149-DM + M222P + L217H + X225M 0.03 BS149-DM + M222P + L217H + X225R 0.02 BS149-DM + M222P + L217H + X225W 0.01

Conclusions: clearly, the mutations at the X225 position have a large effect on the S/H ratio. Many mutations have a superb effect such as with X=N, D, S, C, G and A. Several further enzymes have an over three fold increased S/H ratio as compared to subtiligase (S/H subtiligase=0.9, see example 1) such as with X=L, I, V and T. Also, mutations of X225 into H, Q, and—to a lesser extent—F and E showed an improvement over the wild-type enzyme with X225 being P.

Example 26: Coupling of a Pentapeptide Selectively to the N-Terminus of the A-Chain of Human Insulin

5 mg of human insulin (CAS # 11061-68-0) and 2.5 mg of Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH. TFA were dissolved in 200 μL DMF. Subsequently, 200 μL of phosphate buffer (1 M, pH 8.0) and 200 μL H₂O containing 20 μg of the BS149-DM+M222G mutant were added and the reaction mixture was shaken (150 rpm) at room temperature. After 60 min a 100 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS, showing that 92% of the insulin starting material was converted to a single product, i.e. Ac-Asp-Phe-Ser-Lys-Leu-coupled to the N-terminus of the insulin A-chain.

Example 27: Coupling of a Pentapeptide to the N-terminus of the A- and B-Chain of Human Insulin

5 mg of human insulin (CAS # 11061-68-0) and 5 mg of Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA were dissolved in 200 μL DMF. Subsequently, 200 μL of phosphate buffer (1 M, pH 8.0) and 200 μL H₂O containing 55 μg of BS149-DM+M222G+L217F mutant were added and the reaction mixture was shaken (150 rpm) at room temperature. After 60 min a 100 μL aliquot of the reaction mixture was withdrawn and quenched with 500 μL MSA/water (1/99, v/v) and analyzed by LC-MS, showing that the insulin starting material was completely consumed and converted to three product peaks, i.e. 1) Ac-Asp-Phe-Ser-Lys-Leu-coupled to the N-terminus of the Insulin A-chain (22 area %), 2) Ac-Asp-Phe-Ser-Lys-Leu-coupled to the N-terminus of the insulin B-chain (3 area %) and 3) Ac-Asp-Phe-Ser-Lys-Leu-coupled to the N-terminus of both the Insulin A- and B-chain (75 area %).

SEQUENCES SEQ ID NO 1: wild type gene encoding for subtilisin BPN' amino acids −107 to 275 ENA|K02496|K02496.1 B. Subtilisin BPN' Bacillus amyloliquefaciens GTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGG CGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATAT TGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATTTCT GAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAA ACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAAGATCA CGTAGCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCTGCT CTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATCG ATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCCAGCATGGTTCCTTCTGAAACAAA TCCTTTCCAAGACAACAACTCTCACGGAACTCACGTTGCCGGCACAGTTGCGGCTCTTAAT AACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACGCTGTAAAAGTTCTCG GTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGGGCGATCGCAAA CAATATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTTCTGCTGCTTTAAAAGCG GCAGTTGATAAAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAGGCA CTTCCGGCAGCTCAAGCACAGTGGGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTAGG CGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGCGTAGGACCTGAGCTTGATGTC ATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTGGAAACAAATACGGGGCGTACAACG GTACGTCAATGGCATCTCCGCACGTTGCCGGAGCGGCTGCTTTGATTCTTTCTAAGCACCC GAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGAT TCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTAA SEQ ID NO 2: wild type subtilisin BPN' (mature) >SUBT_BACAM Subtilisin BPN' Bacillus amyloliquefaciens mature 1 to 275 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSUPDLKVAGGASMVPSETNPFQDNNSHGTHVAG TVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVD KAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLP GNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ SEQ ID NO 3: subtilisin BPN' variant with deletion of Ca²⁺ binding loop and S221C and preferably P225 mutation (denoted as P225X) AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAG TVAAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVV VAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNG TCMASXHVAGAAALILSKEIPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ SEQ ID NO 4: subtilisin BPN' variant with preferred mutation positions compared to SEQ ID NO 3 AXXVXYGVXQIKAPALHSQGYTGSNVKVAVXDSGIDSSHPDLXVAGGASXVPSETNPFQDNNSHGTHVAG TVXAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVV VAAAGNXGTSGSSSTVXYPXKYPSVIAVGAVDSSNQRAXFSSVGPELDVMAPGVSIXSTLPGXKYGAXXG TCMASXHVAGAAALILSKHPNWTNTQVRSSLENTXTKLGDSFYYGKGLINVXAAAQ SEQ ID NO 5: The segment of E. coli/B. subtilis shuttle vector pBES: Ptl 149DM His containing the B. subtilis-derived subtilisin (aprE) promoter region (bp 1-197, Takara), the BPN' signal sequence (bp 198-287), the BPN' prodomain (bp 288-518), the mature BS149-DM, 6xHistag, stop codon. From nucleotide 1590 onwards the sequence follows pBES from Takara.   1 ACTAGTGTTC TTTTCTGTAT GAAAATAGTT ATTTCGAGTC TCTACGGAAA TAGCGAGAGA  61 TGATATACCT AAATAGAGAT AAAATCATCT CAAAAAAATG GGTCTACTAA AATATTATTC 121 CATCTATTAC AATAAATTCA CAGAATAGTC TTTTAAGTAA GTCTACTCTG AACTTAAGCA 181 AAAGGAGAGG GACGCGT GTG AGA GGC AAA AAA GTA TGG ATC AGT TTG CTG TTT RBS MluI Val Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe     −107 −105         −100 234 GCT TTA GCG TTA ATC TTT ACG ATG GCG TTC GGC AGC ACA TCC CT GCC Ala Leu Ala Leu Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala −95         −90         −85             −80 282 CAG GCG GCA GGG AAA TCA AAC GGG GAA AAG AAA TAT ATT GTC GGG TTT Gln Ala Ala Gly Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe         −75         −70             −65 330 AAA CAG ACA ATG AGC ACG ATG AGC GCC GCT AAG AAG AAA GAT GTC ATT Lys Gln Thr Met Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile      −60            −55         −50 378 TCT GAA AAA GGC GGG AAA GTG CAA AAG CAA TTC AAA TAT GTA GAC GCA Ser Glu Lys Gly Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala     −45          −40          −35 426 GCT TCA GCT ACA TTA AAC GAA AAA GCT GTA AAA GAA TTG AAA AAA GAC Ala Ser Ala Thr Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp   −30           −25         −20 474 CCG AGC GTC GCT TAC GTT GAA GAA GAT CAC GTA GCA CAC GCG ATG GCG Pro Ser Val Ala Tyr Val Glu Glu Asp His Val Ala His Ala Met Ala −15         −10          −5            1 522 AAG TGC GTG TCT TAC GGC GTA GCG CAA ATT AAA GCC CCT GCT CTG CAC Lys Cys Val Ser Tyr Gly Val Ala Gln Ile Lys Ala Pro Ala Leu His       5             10          15 570 TCT CAA GGC TAC ACT GGA TCA AAT GTT AAA GTA GCG GTT CTT GAC AGC Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Leu Asp Ser      20          25          30 618 GGT ATC GAT TCT TCT CAT CCT GAT TTA AAC GTA GCA GGC GGA GCC AGC Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Ala Gly Gly Ala Ser    35            40          45 666 TTC GTT CCT TCT GAA ACA AAT CCT TTC CAA GAC AAC AAC TCT CAC GGA Phe Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly    50        55      60         65 714 ACT CAC GTT GCC GGC ACA GTT TTG GCT GTT GCG CCA AGC GCA TCA CTT Thr His Val Ala Gly Thr Val Leu Ala Val Ala Pro Ser Ala Ser Leu          70          74* 84 85          90 762 TAC GCT GTA AAA GTT CTC GGT GCT GAC GGT TCC GGC CAA TAC AGC TGG Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp          95           100           105 810 ATC ATT AAC GGA ATC GAG TGG GCG ATC GCA AAC AAT ATG GAC GTT ATT Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn Asn Met Asp Val Ile         110             115         120 858 AAC ATG AGC CTC GGC GGA CCT TCT GGT TCT GCT GCT TTA AAA GCG GCA Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala     125           130           135 906 GTT GAT AAA GCC GTT GCA TCC GGC GTC GTA GTC GTT GCG GCA GCC GGT Val Asp Lys Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala Gly   140          145           150 954 AAC TCT GGC ACT TCC GGC AGC TCA AGC ACA GTG AGC TAC CCT GCT AAA Asn Ser Gly Thr Ser Gly Ser Ser Ser Thr Val Ser Tyr Pro Ala Lys 155          160          165            170 1002 TAC CCT TCT GTC ATT GCA GTA GGC GCT GTT GAC AGC AGC AAC CAA AGA Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln Arg           175           180           185 1050 GCA CCG TTC TCA AGC GTA GGA CCT GAG CTT GAT GTC ATG GCA CCT GGC Ala Pro Phe Ser Ser Val Gly Pro Glu Leu Asp Val Met Ala Pro Gly       190            195          200 1098 GTA TCT ATC TGT AGC ACG CTT CCT GGA GGC AAA TAC GGG GCG CTT TCT Val Ser Ile Cys Ser Thr Leu Pro Gly Gly Lys Tyr Gly Ala Leu Ser     205            210           215 1146 GGT ACG TGC ATG GCA TCT GCG CAC GTT GCC GGA GCG GCT GCT TTG ATT Gly Thr Cys Met Ala Ser Ala His Val Ala Gly Ala Ala Ala Leu Ile      220       225       230 1194 CTT TCT AAG CAC CCG AAC TGG ACA AAC ACT CAA GTC CGC AGC AGT TTA Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln Val Arg Ser Ser Leu 235           240         245           250 1242 GAA AAC ACC GCT ACA AAA CTT GGT GAT TCT TTC TAC TAT GGA AAA GGG Glu Asn Thr Ala Thr Lys Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly         255          260          265 1290 CTG ATC AAC GTA GAA GCG GCA GCT CAG CAC CAC CAC CAC CAC CAC TAA Leu Ile Asn Val Glu Ala Ala Ala Gln His His His His His His ---        270          275          280 1338 AACATAAAAA ACCGGCCTTG GCCCCGCCGG TTTTTTATTA TTTTTCTTCC TCCGCATGTT 1398 CAATCCGCTC CATAATCGAC GGATGGCTCC CTCTGAAAAT TTTAACGAGA AACGGCGGGT 1458 TGACCCGGCT CAGTCCCGTA ACGGCCAAGT CCTGAAACGT CTCAATCGCC GCTTCCCGGT 1518 TTCCGGTCAG CTCAATGCCG TAACGGTCGG CGGCGTTTTC CTGATACCGG GAGACGGCAT 1578 TCGTAATCGG ATGGATCC    BamHI *Deletion with respect to BPN' of amino acid 72-80 (Val-Ala-Ala-Leu-Asn-Asn- Ser-Ile-Gly); GTT GCG GCT CTT AAT AAC TCA ATC GGT. 

What is claimed is:
 1. An enzyme, comprising: mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof, said mutations comprising: a deletion of the amino acids corresponding to positions 75-83; a mutation at the amino acid position corresponding to S221, the mutation being a mutation corresponding to S221C or S221selenocysteine; and a mutation at the amino acid position corresponding to P225; wherein the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2; and wherein the enzyme is a subtilisin BPN′ variant or homologue thereof.
 2. The enzyme of claim 1, wherein the mutation at the amino acid position corresponding to S221 is a mutation corresponding to S221C.
 3. The enzyme of claim 1, comprising one or more mutations, wherein said mutations is/are at an amino acid position corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218, T254 or Q271 of SEQUENCE ID NO
 2. 4. The enzyme of claim 1, wherein said one or more mutations correspond to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A or Q271E.
 5. The enzyme of claim 1, comprising a mutation at both the positions corresponding to N218 and M50.
 6. The enzyme of claim 1, comprising mutations at the amino acid position corresponding to S3C and Q206C, wherein the cysteins at the positions corresponding to position 3 and position 206 form a disulphur bridge.
 7. The enzyme of clam 1, comprising at least six mutations, wherein said mutations correspond to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A or Q271E.
 8. The enzyme of claim 7, wherein the enzyme comprises the mutations corresponding to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A or Q271E of SEQUENCE ID NO
 2. 9. The enzyme of claim 1, comprising one or more mutations at the amino acid position corresponding to N62, G100, 5125, L126, G127, P129, N155, Y217, N218 or M222 of SEQUENCE ID NO
 2. 10. The enzyme of claim 9, comprising a mutation at the position corresponding to M222 of SEQUENCE ID NO
 2. 11. The enzyme of claim 10, wherein said mutation at the position corresponding to M222 is M222G, M222P, M222N, M222E, M222Q or M222A.
 12. The enzyme of claim 1, comprising a mutation at the amino acid position corresponding to Y217 of SEQUENCE ID NO
 2. 13. The enzyme of claim 12, wherein the mutation at the amino acid position corresponding to Y217 is Y217L, Y217N, Y217E, Y217G, Y217F, Y217A, Y217S or Y217H.
 14. The enzyme of claim 13, wherein the mutation at amino acid position corresponding to Y217 is Y217F, Y217G or Y217H.
 15. The enzyme of claim 14, wherein the enzyme comprises mutations at the amino acid positions corresponding to M222 and Y217, wherein the mutations are: M222P and Y217H; M222P and Y217G; M222G and Y217F; or M222G and Y217G.
 16. The enzyme of claim 15, wherein said mutations are M222G and Y217F.
 17. The enzyme of claim 1, comprising at least one mutation selected from the group of mutations at an amino acid position corresponding to Y104, I107, L126, S101, G102, G127, G128, L135 or P168 of SEQUENCE ID NO
 2. 18. The enzyme of claim 17, wherein said mutation is at an amino acid position corresponding to Y104F, Y104S, I107V, I107A, L135N, L135S, L135D or L135A.
 19. The enzyme of claim 1, wherein the mutation at the amino acid position corresponding to P225 is selected from the group consisting of mutations corresponding to P225N, P225D, P225S, P225C, P225G, P225T, P225V, P2251, P225L, P225H and P225Q of SEQUENCE ID NO
 2. 20. The enzyme of claim 1, wherein the enzyme has ligase activity or cyclase activity. 